In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase.

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle.

Review: Environmental impact on early embryonic development in the bovine species.

Assisted reproduction techniques (ARTs) provide access to early stage embryos whose analysis and assessment deliver valuable information. The handling of embryos, including the in vitro production of bovine embryos, is a rapidly evolving area which nonetheless exposes the embryos to unnatural conditions for a period of time. The Fallopian tube provides innumerable quantitative and qualitative factors, all of which guarantee the successful development of the embryo. It is well known that the Fallopian tube can be bypassed, using embryo transfer, resulting in successful implantation in the target recipient animal and the birth of calves. However, the question arises as to whether such circumvention has a negative impact on the embryo during this sensitive development period. First crosstalk between the embryo and its environment confirms mutual recognition activities and indicate bilateral effects. Nowadays, in vitro production of bovine embryos is a well-established technology. However, it is still evident that in vitro generated embryos are not qualitatively comparable to embryos obtained ex vivo. To counteract these differences, comparative studies between in vitro and ex vivo embryos are advantageous, as embryos grown in their physiological environment can provide a blueprint or gold standard against which to compare embryos produced in vitro. Attempts to harness the bovine oviduct were sometimes very invasive and did not result in wide acceptance and routine use. Long-term development and refinement of transvaginal endoscopy for accessing the bovine oviduct has meanwhile been routinely applied for research as well as in practice. Comparative studies combining in vitro development with development in the cattle oviduct revealed that the environmental conditions to which the embryo is exposed before activation of the embryonic genome can have detrimental and lasting effects on its further development. These effects are manifested as deviations in gene expression profiles and methylation signatures as well as frequency of whole chromosomal or segmental aberrations. Furthermore, it was shown that hormonal superstimulation (multiple ovulation and embryo transfer), varying progesterone concentrations as well as metabolic disorders caused by high milk production, markedly affected embryo development in the postpartum period. Assisted reproductive techniques that allow the production and handling of extra numbers of generated embryos promise to have a very high impact on scientific and practical application. Any influence on the early embryonic life, both in animals and in vitro, is accompanied by a sensitive change in embryonic activity and should be assessed in vivo on the basis of physiological conditions before being used for ART.

Meet interesting abbreviations in clinical mass spectrometry: from compound classification by REIMS to multimodal and mass spectrometry imaging (MSI).

This feature article discusses two modern mass spectrometry abbreviations in their clinical applications. Rapid evaporative ionization mass spectrometry (REIMS) is reported as a molecular classification tool useful for spectral features definition prior to mass spectrometry imaging (MSI). REIMS is appreciated not only as an ionization technique coupled with a surgical device but particularly as a biomarker discovery tool. For more complex understanding of pathological processes at cellular and molecular levels, the importance of multimodal approach in imaging applications is documented in the context of fiducial markers needed for hyperspectral data fusion collected by optical microscopy, elemental and molecular MSI. Finally, pathogen inactivation needed prior to the sectioning of the infected tissue is reported, and the impact of formaldehyde crosslinking to signal reduction is discussed.

Protein composition of the phase I Coxiella burnetii soluble antigen prepared by extraction with trichloroacetic acid.

Q fever is a highly infectious, widespread airborne zoonosis caused by Coxiella burnetii bacterium. Humans usually acquire the disease by inhalation of contaminated aerosol produced by infected livestock. Vaccination is the most practical way for prevention and control of the disease in the exposed population. In this work, we reviewed the most important Q-fever outbreaks in Slovakia as well as the progress in vaccine development. One of them represents a soluble antigen complex produced by extraction with trichloroacetic acid from a highly purified C. burnetii phase I strain Nine Mile. It was developed at the Institute of Virology in Bratislava. The protein content of this vaccine was separated by gel electrophoresis and analyzed by mass spectrometry. The study has resulted in the identification of 39 bacterial proteins from which 12 were recognized as immunoreactive. Most of the proteins were involved in bacterium pathogenicity (41.6%) and cell wall maintenance (25%). Four of the immunoreactive proteins may possess the moonlighting activity. Definition of the vaccine components represents a prerequisite for vaccine standardization and approval by governmental authorities.

Lateral resolution of desorption nanoelectrospray: a nanospray tip without nebulizing gas as a source of primary charged droplets.

Desorption nanoelectrospray (nanoDESI) was described in 2007 and it represents a miniaturized version of desorption electrospray without the assistance of the nebulizing gas. Compared to DESI, a nanoelectrospray tip (2 ± 1 µm I.D.) generates primary charged droplets of smaller sizes and lower spray liquid flow rates. This is the first report on utilization of nanoDESI for mass spectrometry imaging (MSI). Its new coupling with a Q-TOF instrument allowed faster mass spectra acquisition (4 Hz) essential for MSI of fine surface details. To evaluate nanoDESI potential for mass spectrometry imaging, etched glass substrates with Rhodamine B patterns of different dimensions were prepared. The Rhodamine B lines were analysed in 1D scanning mode and their width was determined experimentally by nanoDESI measurement. The experimental data revealed that the lateral resolution of nanoDESI is close to 30 µm along the x-axis (orthogonal to the inlet). 2D scanning mode confirmed good resolution along both axes as dye squares with dimensions about 60 µm × 60 µm were easily distinguished. The low flow rate of the spray liquid reduced undesirable analyte washing effects, which allowed repeated scanning analysis of the surface. The presented results demonstrate the applicability of nanoDESI for high surface resolution mass spectrometry imaging.

Role of the oviduct in early embryo development.

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows.

The aim of this study was to examine the direct effect of lactation on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one primiparous Holstein heifers were used. Immediately after calving, half of the cows were dried off (i.e., never milked), and the other half entered the milking herd and were milked twice daily. Jugular blood samples were taken twice per week from 15 d before calving to approximately 100 d postpartum to measure nonesterified fatty acids, ß-hydroxybutyrate, glucose, insulin, and insulin-like growth factor-I. At the same time, body weight and body condition score were recorded for each cow. At approximately 60 d postpartum (experiment 1), approximately 65 two- to four-cell embryos, produced by in vitro maturation and fertilization, were endoscopically transferred to the oviduct ipsilateral to the corpus luteum of all cows on d 2 of the estrous cycle. Five days later (d 7), the oviduct and uterus were flushed nonsurgically and the number of embryos developing to the blastocyst stage was recorded. At approximately 90 d postpartum (experiment 2), the estrous cycles of the same cows were resynchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterus of each recipient on d 7. All cows were slaughtered on d 14 to assess embryo survival and dimensions. Body weight and body condition score were significantly different between groups for the entire postpartum period of the study. Concentrations of nonesterified fatty acids and ß-hydroxybutyrate were higher and concentrations of glucose, insulin, and insulin-like growth factor-I were lower in lactating compared with nonlactating cows. Embryo recovery rates from lactating and dry cows were similar. In experiment 1, fewer embryos developed to the blastocyst stage in the lactating cows compared with the nonlactating cows. In experiment 2, embryo survival and conceptus dimensions were not different between lactating and nonlactating cows. In conclusion, the data indicate that the reproductive tract of the lactating dairy cow is compromised in its ability to support early embryo development compared with that of matched dry cows and this may contribute to early embryo mortality observed in such animals.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction.

BACKGROUND: In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS: Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS: The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS: The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Transcriptomic analysis of in vivo and in vitro produced bovine embryos revealed a developmental change in cullin 1 expression during maternal-to-embryonic transition.

Pre-implantation embryos derived by in vitro fertilization differ in their developmental potential from embryos obtained in vivo. In order to characterize changes in gene expression profiles caused by in vitro culture environment, we employed microarray constructed from bovine oocyte and preimplantation embryo-specific cDNAs (BlueChip, Université Laval, Québec). The analysis revealed changes in the level of 134 transcripts between in vitro derived (cultured in COOK BVC/BVB media) and in vivo derived 4-cell stage embryos and 97 transcripts were differentially expressed between 8-cell stage in vitro and in vivo embryos. The expression profiles of 7 selected transcripts (BUB3, CUL1, FBL, NOLC1, PCAF, GABPA and CNOT4) were studied in detail. We have identified a switch from Cullin 1-like transcript variant 1 to Cullin 1 transcript variant 3 (UniGene IDs BT.36789 and BT.6490, respectively) expressions around the time of bovine major gene activation (8-cell stage). New fibrillarin protein was detected by immunofluorescence already in early 8-cell stage and this detection correlated with increased level of fibrillarin mRNA. The qRT-PCR analysis revealed significant differences in the level of BUB3, NOLC1, PCAF, GABPA and CNOT4 gene transcripts between in vivo derived (IVD) and in vitro produced (IVP) embryos in late 8-cell stage. The combination of these genes represents a suitable tool for addressing questions concerning normal IVD embryo development and can be potentially useful as a marker of embryo quality in future attempts to optimize in vitro culture conditions.

Assessment of actin cytoskeleton and nuclei in bovine blastocysts developed under different culture conditions using a novel computer program.

This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro-to-in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir-sourced ovaries were matured in vitro and allocated to four groups: IVP-group embryos developed up to blastocyst stage in vitro. Gamete intra-fallopian transfer (GIFT)-group oocytes were co-incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2-7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)-group embryos were obtained by superovulating and inseminating heifers; the heifers' genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2-7 transfer did not differ significantly from each other. Gamete intra-fallopian transfer- and MOET-group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment.

Effect of elevated circulating progesterone concentration on bovine blastocyst development and global transcriptome following endoscopic transfer of in vitro produced embryos to the bovine oviduct.

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F(2 alpha) analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows.

Infertility in dairy cattle is a multifactorial problem that may be linked to follicle development and the quality of the ovulated oocyte, to sperm transport and fertilization, to the reproductive tract environment, or to a combination of these factors. Using a state-of-the-art endoscopic embryo transfer technique, the aim of this study was to compare the ability of the reproductive tract of postpartum dairy cows and nulliparous heifers to support the development of early embryos to the blastocyst stage. Bovine embryos of 2 to 4 cells (n=1,800) were produced by in vitro maturation and fertilization of oocytes derived from the ovaries of slaughtered cattle. The estrus cycles of nulliparous Holstein heifers (n=10) and postpartum Holstein cows (n=8, approximately 60 d postpartum) were synchronized using an 8-d controlled internal drug release device coupled with prostaglandin injection. On d 2, one hundred 2- to 4-cell embryos were endoscopically transferred to the oviduct ipsilateral to the corpus luteum. Five days later, on d 7, the oviduct and uterus were flushed nonsurgically to recover the embryos. The number of embryos developing to the blastocyst stage was recorded immediately at recovery and following overnight culture in vitro. A representative number of blastocysts from heifers and cows were stained to assess cell number. Progesterone concentrations were lower in cows than in heifers on d 5, 6, and 7 (d 7=2.39+/-0.33 vs. 5.34+/-0.77ng/mL, respectively). More embryos were recovered from heifers than cows (79.0+/-7.0 vs. 57.2+/-11.4%). Of the embryos recovered, 33.9+/-3.6% had developed to the blastocyst stage in the heifer oviduct compared with 18.3+/-7.9% in the postpartum cow oviduct. There was no evidence of a difference in blastocyst quality as evidenced by total cell number in the blastocysts (71.2+/-5.7 vs. 67.0+/-5.3, respectively). In conclusion, the reproductive tract of the postpartum lactating dairy cow may be less capable of supporting early embryo development than that of the nonlactating heifer, and this may contribute to the lower conception rates observed in such animals.

Endoscopic approaches to manage in vitro and in vivo embryo development: use of the bovine oviduct.

The oviduct plays a major part in different reproductive processes providing the microenvironment for numerous steps in early embryogenesis. Consequently, there is a growing demand to perform comparative studies focusing on causal mechanisms related to embryo development within its environment including complex and holistic strategies. However, the routine flushing and transfer procedure of bovine embryos is limited to the morula and blastocyst stage. Additionally, the use of in vitro production of bovine embryos provides access to an extra amount of embryos at various stages. But the quality of these embryos does not reflect the quality of its ex vivo counterparts. For two decades our own studies have focused on use of the oviductal environment of different species to optimize early embryo development for different purposes. The current article briefly highlights some main characteristics of the fallopian tube and reviews the endoscopic approach to access the fallopian tube using the stepwise minimal invasive technique established in different species.

The effect of long-term in vivo culture in bovine oviduct and uterus on the development and cryo-tolerance of in vitro produced bovine embryos.

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short- vs long-term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP-Group), or after IVF and 2-3 days of IVC, 4-8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo-Group) or IVM oocytes were co-incubated with spermatozoa for 3-4 h and transferred into the oviducts of synchronized heifers (GIFT-Group). Embryos of the In Vivo-Group and the GIFT-Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa-medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP-Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo-survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo-tolerance. However, the duration of in vivo culture crucially influenced the cryo-tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.

Expression of apoptosis regulatory genes and incidence of apoptosis in different morphological quality groups of in vitro-produced bovine pre-implantation embryos.

Apoptosis occurs during early development in both in vivo- and in vitro-produced embryos, and is considered as one of the causes of embryonic loss. The objectives of this study were, therefore, investigating stage-specific expression profiles of apoptosis regulatory genes in three quality groups of in vitro-produced bovine pre-implantation embryos; and analysing the relationship between cell number and DNA fragmentation with expressions of those genes. The relative abundance of mRNA of 9 pro- (Bax, caspase-9, Bcl-xs, P53, Caspase-3 and Fas) and anti- (Bcl-w and Mcl-1) apoptotic genes was analysed. Differential cell staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling were performed to analyse the variation in cell numbers and detect apoptotic nuclei respectively. Expression of Bax and Caspase-3 genes was significantly (p < 0.05) higher in poor quality pre-implantation embryos as compared with that of morphologically good quality embryos of the same developmental stages. Moreover, Mcl-1 expression was significantly higher in good quality immature oocytes than that in the poor quality group. Moreover, higher DNA fragmentation was evidenced in morphologically poor quality blastocysts. In conclusion, our study demonstrates that Bax, caspase-3 and Mcl-1 can be used as potential markers of embryo quality to evaluate in vitro-produced bovine embryos. Further studies are required to investigate specific molecular signatures that can be used in evaluating in vivo-derived embryos.

[Long-term results of use of the CLS stem in primary total hip arthroplasty]. / Dlouhodobé výsledky CLS dríku u primární náhrady kycle.

PURPOSE OF THE STUDY: The aim of the study was to evaluate the results of primary total hip arthroplasty with the use of the CLS stem at 11 to 17 years after implantation. MATERIAL AND METHODS: A total of 108 patients (122 hips) in whom a CLS stem was used in the 1991-1996 period were evaluated. The group included 34 men and 74 women, with an average age of 48 years (range, 28-63). The CLS stem with a neck-shaft angle of 145 degrees and the CLS expansion cup were used in all patients. Clinical outcomes were evaluated by Merle d'Aubigné-Postel score and Harris hip score, radiological examination was completed on AP and lateral views of the pelvis and the operated hip. RESULTS: The average follow-up was 16.4 years (range, 11-17). The average Merle d'Aubigné-Postel score was 14.5 (range, 13.9-17.0) points and the average Harris hip score was 84.8 (range, 70-99) points. Very good or good outcomes were found in 81% of the patients. Three patients underwent revision surgery, in one for septic loosening, in one for aseptic loosening and in one for varus stem position leading to instability. The radiographs evaluated as described by Engh showed 116 stable stems, three fibrous stable and three unstable stems. Subsidence of more than 3 mm, without any further deterioration, was found in five hips at 12 months post-operatively. Seven hips showed one radiolucent line, four showed two radiolucent lines and three hips showed three radiolucent lines, all of them being less than 2 mm wide. DISCUSSION: Radiographic evidence of a stable stem in 116 hips (116/122) suggests a high reliability of the implant. Assessment of radiolucent lines showed 108 hips without radiographic demarcation and 11 hips with slight demarcation. The signs of stress-shielding grade 1 were found in 28 hips. Good results of arthroplasty with the CLS stem can be attributed to its three-dimensional wedge-shaped design that allows for an optimal press-fit in the metaphyseal region. The porous surface provides reliable osteointegration. Stress-shielding is prevented by optimal stress distribution in the metaphyseal region and by a modulus of elasticity of titanium alloy which closely approximates the modulus of elasticity of bone. CONCLUSIONS: At 15 years post-operatively, the cumulative probability of clinical survivorship of the CLS stem was 98.3 %, and cumulative probability of radiographic survivorship was 87.7 %. The advantages of this stem include a technically simple implantation, reliable osteointegration and long-term stability even in high demanding patients.

[Mathematical simulation of stem/cement/bone mechanical interactions for Poldi-Cech, CF-30, MS-30 and PFC femoral components]. / Výpoctové modelování mechanických interakcí dríku Poldi-Cech, CF-30, MS-30 a PFC s cementem a kostní tkání

PURPOSE OF THE STUDY The mid-term longevity of femoral components varies considerably, with some showing failure due to early aseptic loosening. Since the hip joint is subject to heavy mechanical loads, it can be assumed that the mechanical interaction of the implant, bone cement and femur will play a key role in the resultant reliability of an arthroplasty. This study was designed to examine this mechanical interaction in four femoral components different in construction (Poldi-Cech, CF-30, MS-30 and PFC) using mathematical simulation. MATERIAL AND METHODS Four stem/cement/femur 3-D mathematical models, comparable in quality, infolving the Poldi-Cech, CF-30, MS-30 and PFC stems, respectively, were constructed. A 3-D model for each stem was created according to its real, middle-size femoral component. Each 3-D model of the cement mantle corresponded in shape to the mantle of the appropriate real stem, with its thickness based on the recommended values of 4-7 mm in the proximal and 1-3 mm in the distal part, and with the cement mantle reaching as far as 10 mm distal to the femoral stem tip. For simplicitys sake the outer surface of the cement mantle was simulated as smooth. A 3-D model involving the proximal epiphysis and the metaphysis of a femur was reconstructed, based on a series of CT cross-sections obtained periodically at 10.5-mm and 2.5-mm distances. The sten/cement/femur model with the MS-30 stem also included a centraliser. The mechanical interaction of the stem, bone cement and bone tissue was examined by means of mathematical stimulation using ANSYS 5.7 software based on finite element analysis. RESULTS For the sake of simplicity, only two key parameters are presented, namely, contact stress at the stem-cement interface and equivalent deformation in the stem/cement/femur system. The least satisfactory stress loading was in the CF-30 stem whose sharp edges showed the values of contact stress about six-times higher than on the mid-medial portion of the stem, with the sharp edges behaving as stress concentrators. A satisfactory stress loading was found in Poldi-Cech, MS-30 and PFC stems, in which contact stress was evenly distributed along the whole lenght of the stem and the values at the edges and on the midmedial portion did not differ much. DISCUSSION The distribution of contact stress is one of the most important factors for the long-term longevity of implants. It was found least satisfactory in the CF-30 stem whose sharp edges act as stress condenser adversely affecting not only the stemcement interface, but also the resultant stress distribution within the femur. The most satisfactory results of stress distribution were recorded in the Poldi-Cech and MS-30 stems. The PFC stem also responded satisfactorily to the simulated stress loading. However, on loading whose substantial part would be torsion, the stems circular or oval cross-section could interfere with rotation stability of the implant; but this was impossible to detect by the mathematical simulation used in this study. CONCLUSIONS The results presented here show that, in the Poldi-Cech, CF-30, MS-30 and PFC femoral stems, a good agreement was achieved between the results of their clinical application and those of mathematical modelling of their mechanical properties. It can be concluded that mechanical interaction among the femoral stem, cement mantle and bone tissue plays the key role in the long-term longevity of such an implant. Key words: Poldi-Cech, CF-30, MS-30, PFC, mechanical interaction, contact stress.

Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo.

Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.

Endoscopic recovery of early preimplantation bovine embryos: effect of hormonal stimulation, embryo kinetics and repeated collection.

The collection of extra numbers of bovine embryos by superstimulation of donors underlies variation concerning yield of morulae and blastocysts. Our study aimed at establishing a correlation between hormonal treatment and embryo development during oviductal passage including repeated flushing. A transvaginal endoscopic procedure was used to flush the oviducts at six different time intervals (beginning at 24 h until 105 h) after artificial insemination. In total, 119 animals were superovulated using either FSH or eCG. The hormonal treatment resulted in the stimulation of 2076 follicles of which 77% (1590 CL) ovulated. The bilateral flushing resulted in the collection of 1411 complexes (collection rate: 89%), of which 78% (1098) were assessed as viable embryos. The use of FSH resulted in significantly more stimulated follicles and ovulation sites compared with eCG (p < 0.001). Generally, the embryo kinetics were similar among the FSH and eCG treated animals. However, the embryo cleavage of the eCG treated animals was ahead of that of the FSH group comparing the different collection time points. The overall proportions of non-viable embryos in both groups were similar. Regarding the embryo collection intervals in the eCG group, this proportion significantly increased during 51-105 h compared to 24-50 h (p < 0.05), whereas FSH delivered constant results. It was shown that the repeated endoscopic collection of oviductal stage embryos had no negative influence on the collection parameters. It is concluded that the introduced transvaginal endoscopic technique could have main impact on further studies focusing on early embryo development.

[Surgical management of hallux valgus by techniques preserving the first metatarsophalangeal joint: long-term results]. / Dlouhodobé výsledky operacního resení hallux valgus technikami zachovávajícími I. metatarzofalangeální kloub.

PURPOSE OF THE STUDY: Hallux valgus is a frequent static deformity of feet in shoe-wearing populations. Lasting problems usually require surgical management. The authors evaluate the long-term results of such treatment by either McBride's operation or chevron osteotomy, or by combination of both. MATERIAL: A group of 72 patients with hallux valgus underwent 84 operations, with the use of McBride's procedure, chevron osteotomy or a combination of both, at the First Department of Orthopedic Surgery, St. Anne's Teaching Hospital in Brno, in the years 1993-1995. At 10-year follow-up they were evaluated on the basis of patients' subjective satisfaction and the degree of correction measured by hallux valgus angle (HVA) and intermetatarsal angle (IMA). METHODS: Surgery is carried out under general or spinal anesthesia, with application of a pneumatic tourniquet, after a standard preparation of the operating field. In the modified chevron osteotomy, a "V"-shaped osteotomy of the distal metatarsal is created (V-osteotomy angle is 70 to 80 degrees), which allows the first metatarsal head to be shifted laterally. The modified McBride's procedure is based on transposition of the adductor hallucis tendon onto the first metatarsal head; lateral sesamoidectomy may be necessary. A combination of both techniques involves V-shaped osteotomy of the first metatarsal bone and transposizion of the adductor hallucis tendon, with lateral sesamoidectomy, when necessary. These surgical procedures always include excision of a bursa at the first metatarsal head, removal of a medial eminence of the first metatarsal head and lateral capsulotomy of the first metatarsophalangeal joint. The authors evaluated: 1) the degree of correction by comparing the HVA and IMA on pre-operative radiographs with those measured at 10 years after surgery; 2) subjective satisfaction of the patients who received a questionnaire asking about big-toe position, pain, problems associated with footwear, sores over the metatarsophalangeal joint of the big toe and mobility of this joint. RESULTS: Of the patients undergoing chevron osteotomy (20 procedures), 95 % reported satisfaction; the mean degree of correction was 13 degrees for HVA and 4 degrees for IMA. Of the patients undergoing McBride's procedure (45 operations), 60 % were satisfied; this group had the lowest mean degree of correction, i. e., 4.8 degrees for HVA and -0.6 degrees for IMA. Of the patients undergoing the combined technique (19 operations), 74 % reported satisfaction and the mean degree of correction was highest, i. e., 17.9 and 4.5 degrees for HVA and IMA, respectively. Two patients of this group developed hallux varus, but their HVA and IMA values were not included in the assessment because they would adversely affect the objective evaluation of all the patients. However, in the subjective evaluation of the whole group, these two unsatisfied patients were included. DISCUSSION: In agreements with the majority of published results, the authors conclude that a higher correction is achieved with chevron osteotomy than with McBride's operation. Subjective satisfaction reported in the literature is not consistent, but it is either similar in both procedures or better in chevron osteotomy. In this study, chevron osteotomy resulted in high patient satisfaction (95 %), good correction (HAV, 13 degrees ; IMA, 4 degrees ) and a minimum of complications. McBride's procedure resulted in the lowest correction (HAV, 4.8 degrees ; IMA, -0.6 degrees ) as well as the lowest satisfaction (60 %). Our results show that younger patients (up to about 35 years) responded with better outcomes. The combined method achieved the highest degree of correction (HAV, 17.9 degrees ; IMA, 4.5 degrees ) and 74 % satisfaction, but was associated with the risk of hallux varus development. CONCLUSIONS: If indication criteria are respected, surgical procedures are competently performed and good post-operative care is provided, it is not necessary to combine the operation techniques in order to achieve good long-lasting correction and patients' satisfaction.