Detection Trend of Helminth Eggs in the Strata Soil Samples from Ancient Historic Places of Korea.

For several years, we have conducted a series of studies on the patterns of ancient parasitism prevailing in the soil of rural and urban areas of past Kingdom of Korea. Actually, during our survey of paleoparasitology in archaeological sites of Korean peninsula, numerous ancient parasite eggs were discovered in the samples from the city districts of Hansung (Joseon) and Buyeo (Baikje), the palace moat at Gyeongju (Silla), shell-midden site at Bonghwang-dong (Silla to Joseon), and the reservoir found in Hwawangsansung fortress (Silla). By the paleoparasitological studies, with respect to parasitism in the high-density populations of ancient towns and cities, we have managed to catch glimpses of the patterns prevalent therein: a serious parasitic contamination of the soil in ancient urban areas, but not in rural areas of the past. Our historical research also proposed the plausible mechanism of parasite infection very serious indeed among urban populations in Korean history. Although city dwelling doubtless has accrued significant benefits for people and populations with agriculture, it can be equally supposed that living in such highly populated areas might have facilitated the spread of parasite infection.

Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs.

Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests with fully exposed eggs only became widespread among Euornithes. A potential co-evolution of open nests and brooding behavior among maniraptorans may have freed theropods from the ground-based restrictions inherent to covered nests and allowed the exploitation of alternate nesting locations. These changes in nesting styles and behaviors thus may have played a role in the evolutionary success of maniraptorans (including birds).

DNA barcoding of freshwater ichthyoplankton in the Neotropics as a tool for ecological monitoring.

Quantifying and classifying ichthyoplankton is one of the most effective ways of monitoring the recruitment process in fishes. However, correctly identifying the fish based on morphological characters is extremely difficult, especially in the early stages of development. We examined ichthyoplankton from tributaries and reservoirs along the middle stretch of the Paranapanema River, one of the areas most impacted by hydroelectric projects in the Neotropics. Matching DNA sequences of the COI gene (628-648 bp) allowed us to identify 99.25% of 536 samples of eggs (293) and larvae (243) subjected to BOLD-IDS similarity analysis with a species-level threshold of 1.3%. The results revealed 37 species in 27 genera, 15 families and four orders, some 23.8% of documented fish species in the Paranapanema River. Molecular identification meant that we could include data from egg samples that accounted for about 30% of the species richness observed. The results in this study confirm the efficacy of DNA barcoding in identifying Neotropical ichthyoplankton and show how the data produced provide valuable information for preparing plans for conserving and managing inland waters.

Soil contamination by parasite eggs in rural village in the Philippines.

Infectious diseases caused by soil-transmitted helminths (STHs) are important diseases of humans, which affect about one third of the world's population. Examination of soil can be used to estimate the risk of STH infection in humans. We carried out this survey to clarify the current status of soil contamination by parasite eggs and to assess the risk of STH infection. During survey periods, we examined soil, faeces, and the lifestyle of residents. Six genera and eight species of parasite eggs including Ascaris lumbricoides, Toxocara cati, Toxocara canis, and Trichuris trichiura were recovered from 85 out of 120 soil samples (71%). Contamination of soil by parasite eggs had spread widely throughout the village, and 50% of eggs recovered had already developed into fertilized eggs. It is remarkable that Ascaris eggs were recovered from inside the houses. Prevalence of STH in school children was 63%. This may indicate that school or preschool children cause soil contamination. Some of the eggs recovered were not only from humans but also from dogs and cats. From the results obtained, the need for health education with regards to zoonoses was revealed because 77% of fertilized Toxocara spp. eggs were detected. We conclude that the risk of STH infection in residents was extremely high, because the soil in this village was highly contaminated by infective parasite eggs.

Morphology and identification of fly eggs: application in forensic entomology.

Fly eggs found in corpses can be used as entomological evidence in forensic investigation. This study aims to investigate the morphology of forensically important fly eggs. Eggs of Chrysomya rufifacies, Chrysomya megacephala, Chrysomya pinguis, Chrysomya nigripes, Hypopygiopsis tumrasvini, Lucilia cuprina, Lucilia porphyrina and Musca domestica were examined using 1% potassium permanganate solution for 1 min. Morphometric analysis revealed that the mean length of Hy. tumrasvini (1.63 mm) and C. pinguis (1.65 mm) eggs was the longest, followed by that of L. porphyrina (1.45 mm), C. rufifacies (1.34 mm). The egg length, width of median area and darkness staining of hatching pleats were distinctive features. Four categories of median area were proposed, based on width; (1) distinctly wide (Megaselia scalaris, Synthesiomyia nudiseta); (2) wide (C. nigripes, M. domestica); (3) slightly widening (Hy. tumrasvini, L. cuprina, L. porphyrina); and (4) narrow (C. rufifacies, C. albiceps, C. megacephala, C. pinguis). Four species were examined using SEM, i.e., C. megacephala, C. pinguis, Hy. tumrasvini and L. porphyrina. The eggs of C. megacephala demonstrated swollen hatching pleats. Inside, the hexagon of the chorion appeared as a sponging bumpy feature. The egg of C. pinguis was similar to C. megacephala, except for the sponging bumpy feature on the outer surface of the hatching pleats. Regarding Hy. tumrasvini and L. porphyrina, their island structure was apparent at the inner surface of the upright hatching pleats. The key for identifying these eggs together with other reported species in Thailand has been updated.

Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacillaflava), with description of in vitro ookinetes of Haemoproteus motacillae.

In studies of haemosporidian systematics and taxonomy, a combination of microscopic examination and molecular identification has been recommended. The yellow wagtail (Motacillaflava) is a frequently used species in studies of host-parasite interactions; blood parasites of this bird have been well studied using microscopic examination of blood films, but data on molecular characterization of the parasites are scattered. Here we present the first study linking several haemosporidian cyt b sequences with their morphospecies parasitizing the yellow wagtail, combining light microscopy and nested PCR methods. The lineage hYWT1 of Haemoproteus motacillae was the most prevalent parasite; it was recorded in 38% of the birds. The latter lineage and the lineages hYWT2, hYWT3, hYWT5 and hYWT6 form a well-supported clade on the phylogenetic tree and likely represent intraspecific genetic variation of H. motacillae, with genetic divergence of 0.3 - 1.5 % among these lineages. Microscopic examination of smears prepared during an in vitro experiment, which was designed for observation of developing ookinetes, showed that H. motacillae ookinetes were present 5 hours after exposure of blood to air at 18-20° C. Ookinetes of this parasite belong to a group of large Haemoproteus spp. ookinetes (19.9 µm in length on average). Illustrations of the parasites are given. Two of reported Plasmodium lineages, pCOLLI and pYWT4 are phylogenetically closely related to the widespread host generalist parasites Plasmodium relictun (cyt b lineages pSGS 1, pGRW4 and pGRW11), but it was not possible to establish this morphologically. This study contributes to the molecular identification of avian haemosporidian parasites and provides information on morphology of H. motacillae ookinetes, which is additional information assisting the microscopic identification of this species.

Comparative morphology of minute intestinal fluke eggs that can occur in human stools in the republic of Korea.

The egg morphology of minute intestinal flukes (MIF) that can occur as human infections in the Republic of Korea, i.e., Metagonimus yokogawai, M. miyatai, M. takahashii, Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Stictodora fuscata, Pygidiopsis summa, and Gymnophalloides seoi, was studied in comparison with Clonorchis sinensis. The adult worms were obtained from residents of endemic areas, and their intrauterine eggs were studied and measured using light microscopy; the length, width, length-width ratio (LWR), and Faust-Meleney index (FMI). Several specimens were processed for scanning electron microscopy (SEM), and before gold-coating, the uterine portion of each fluke was etched with a sharp pin in order to expose the eggs. The MIF eggs were ovoid, pyriform, or elliptical with a size range of 21-35×12-21 µm. S. fuscata eggs revealed the highest FMI (largest in the area) and lowest LWR, whereas P. summa eggs showed the lowest FMI and medium LWR. SEM revealed that G. seoi and S. fuscata had remarkably clean shell surface lacking the muskmelon-like structure which is prominent in C. sinensis eggs. In Metagonimus spp., H. continua, H. nocens, and S. falcatus eggs, minute surface ridges were recognizable though less prominent compared with C. sinensis. On the surface of P. summa eggs, thread-like curly structures were characteristically seen. The results revealed that important differential keys for MIF eggs include the length, width, area (FMI), shape of the eggs, and the extent of the muskmelon-like structure or ridges on their shell surface and operculum.

Comparative Morphology of Minute Intestinal Fluke Eggs That Can Occur in Human Stools in the Republic of Korea

The egg morphology of minute intestinal flukes (MIF) that can occur as human infections in the Republic of Korea, i.e., Metagonimus yokogawai, M. miyatai, M. takahashii, Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Stictodora fuscata, Pygidiopsis summa, and Gymnophalloides seoi, was studied in comparison with Clonorchis sinensis. The adult worms were obtained from residents of endemic areas, and their intrauterine eggs were studied and measured using light microscopy; the length, width, length-width ratio (LWR), and Faust-Meleney index (FMI). Several specimens were processed for scanning electron microscopy (SEM), and before gold-coating, the uterine portion of each fluke was etched with a sharp pin in order to expose the eggs. The MIF eggs were ovoid, pyriform, or elliptical with a size range of 21-35x12-21 microm. S. fuscata eggs revealed the highest FMI (largest in the area) and lowest LWR, whereas P. summa eggs showed the lowest FMI and medium LWR. SEM revealed that G. seoi and S. fuscata had remarkably clean shell surface lacking the muskmelon-like structure which is prominent in C. sinensis eggs. In Metagonimus spp., H. continua, H. nocens, and S. falcatus eggs, minute surface ridges were recognizable though less prominent compared with C. sinensis. On the surface of P. summa eggs, thread-like curly structures were characteristically seen. The results revealed that important differential keys for MIF eggs include the length, width, area (FMI), shape of the eggs, and the extent of the muskmelon-like structure or ridges on their shell surface and operculum.

Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization.

Before fertilization, the genome packaging of male and female gametes is very different. Indeed, whereas the female haploid genome is associated with histones in a somatic-like chromatin structure, most of the male genome is tightly bound to protamines. However, it has recently been demonstrated that the pericentric heterochromatin regions of the male genome are associated with specific H2A-like histone variants, named H2AL1 and H2AL2, suggesting a heterogeneous organization. The fate and role of the sex-specific genome packaging transmitted by germinal cells to the embryo are not well understood. The aim of the present study was to follow reprogramming of the parental genomes in early embryos after in vivo fertilization. We show here that two typical epigenetic markers, trimethylated lysine 9 of histone H3 (TriMethylH3K9) and acetylated H4, are asymmetrically distributed between the parental genomes in one-cell mouse embryos, confirming data from embryos obtained after intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Indeed, whereas the maternal genome is highly enriched with trimethylH3K9, this mark is not detected in the paternal genome. On the contrary, histone H4 incorporated in the paternal genome is highly acetylated at an early stage, while in the maternal pronucleus, the level of acetylated H4 remains low in early one-cell embryos and becomes enriched at a later stage. Moreover, our results suggest a very quick disappearance of histone H2A variants H2AL1 and H2Al2 from the paternal pericentric heterochromatin regions after sperm-egg fusion.

Identification of fly eggs using scanning electron microscopy for forensic investigations.

Forensic entomology is the science that studies the role of insects in decomposing corpses and one of the most common uses is to estimate the post-mortem interval (PMI) based on insect activity on a decomposing body. Usually, flies are the first insects to reach a carcass and are able to oviposit on carrion within a few hours after death. Scanning electron microscopy (SEM) gives detailed information about morphological characters helping to identify the immature forms of flies and consequently serves as a tool in crime scene investigations. Sometimes, only eggs and larvae are found in corpses. Some dipteral species are important because their larvae develop in organic matter. The aim of this study is to identify eggs of species of forensic importance, such as Chrysomya megacephala, Chrysomya putoria, Lucilia cuprina, Lucilia eximia and Ophyra aenescens, using scanning electron microscopy (SEM). C. megacephala had no anastomosis or holes at the top of the islands and C. putoria had few anastomoses and no holes, whereas L. eximia and O. aenescens were found to have anastomoses and holes and L. cuprina had only anastomoses. The median area was bifurcated anteriorly in C. megacephala, L. eximia and O. aenescens and rounded in C. putoria and L. cuprina. Also the sculptures observed in the chorionic cells, the length and the way that median area ends up posteriorly are characteristics of great diagnostic value to identify muscoids of forensic importance.

Bayesian classification for the selection of in vitro human embryos using morphological and clinical data.

In vitro fertilization (IVF) is a medically assisted reproduction technique that enables infertile couples to achieve successful pregnancy. Given the uncertainty of the treatment, we propose an intelligent decision support system based on supervised classification by Bayesian classifiers to aid to the selection of the most promising embryos that will form the batch to be transferred to the woman's uterus. The aim of the supervised classification system is to improve overall success rate of each IVF treatment in which a batch of embryos is transferred each time, where the success is achieved when implantation (i.e. pregnancy) is obtained. Due to ethical reasons, different legislative restrictions apply in every country on this technique. In Spain, legislation allows a maximum of three embryos to form each transfer batch. As a result, clinicians prefer to select the embryos by non-invasive embryo examination based on simple methods and observation focused on morphology and dynamics of embryo development after fertilization. This paper proposes the application of Bayesian classifiers to this embryo selection problem in order to provide a decision support system that allows a more accurate selection than with the actual procedures which fully rely on the expertise and experience of embryologists. For this, we propose to take into consideration a reduced subset of feature variables related to embryo morphology and clinical data of patients, and from this data to induce Bayesian classification models. Results obtained applying a filter technique to choose the subset of variables, and the performance of Bayesian classifiers using them, are presented.

Biotechnological and clinical outcome of in vitro fertilization in non-obese patients with polycystic ovarian syndrome.

INTRODUCTION: Polycystic ovarian syndrome (PCOS) is a hormonal and metabolic disorder which poses problems with controlled ovarian stimulation (COH). It has been also postulated that PCOS patients have oocytes and embryos with poorer quality which affects IVF results. AIM: To verify IVF outcome in non-obese patients with PCOS. MATERIALS AND METHODS: IVF results of 71 non-obese PCOS patients with 243 non-obese non-POCS patients, regardless of stimulation protocol, from years 2004-2006 were compared. RESULTS: Biotechnological results of PCOS patients in opposition to non-PCOS patients were respectively as follows: higher average number (10.19 vs. 7.61; p=0.001) and percentage (82.34% vs. 76.25%; p=0.025) of retrieved mature M2 oocytes; similar (77.01% vs. 76.75%; p=0.835) fertilization rate with higher average number of embryos (7.633 vs. 5.650 p=0.003); higher average number (4.830 vs. 3.304; p=0.001) and percentage (65.66% vs. 60.57%; p=0.006) of embryos with optimal Z1 and Z2 pronuclei pattern according to Scott; higher average number of class Aembryos (3.57 vs. 2.34; p=0.001). Similar number of embryos were transferred in both groups (2.408 vs. 2.485, p=0.552). Clinical results in PCOS and non-PCOS patients were as follows: similar stimulation duration (10.53 days vs. 10.31 days; p=0.639) with significant less gonadotropin total usage (1866.54 IU vs. 2276.18 IU; p=0.001). Also clinical pregnancy per transfer (57.75% vs. 41.98%; p=0.021) and delivery per transfer (45.07% vs. 32.51%; p=0.066) were more often in PCOS patients with comparable miscarriages (12,68% vs. 6,58%; p=0.131) and ectopic pregnancy (0.00% vs. 2.06%; p=0.591) rates, respectively. CONCLUSION: PCOS in non-obese patients is linked with good biotechnological and clinical IVF outcome.

Pronuclear scoring as a predictor of embryo quality in in vitro fertilization program.

Many strategies have been proposed for the selection of viable embryos for transfer in human assisted reproduction. These have included morphological scoring criteria for 20, 28, 44 and 68 h after insemination. The embryo selection is based on morphology, degree of fragmentation and development to the 8-cell. All have shown some correlation with implantation. However, the overall success of these methods is still limited, with over 50% of all transferred embryos failing to implant. Pronuclear zygote morphology has gained much attention recently due to its positive value in predicting implantation and pregnancy. This prospective study involved 178 conventional IVF patients only. The key aspects of pronuclear scoring and namely the presence of a cytoplasmic halo were related to day 3 of development and morphology in a retrospective study. The Z-score and the presence/absence of a halo had significant effect on the rate of development on day 3 embryo. Low Z-score result in slow development and poor morphology. The absence of a halo also resulted in slow and poor development, low morphology, increased fragmentation.

Physical growth charts from birth to six years of age in Japanese twins.

BACKGROUND: The purpose of this study was to analyze the characteristics of the physical growth of twins in childhood and to present growth charts of Japanese twins. METHODS: The subjects consisted of 2029 pairs of normally developed Japanese twins. Growth data were obtained by mailed or hand-distributed questionnaires. Factors that affect body weight and height/length at selected ages were analyzed by stepwise regression analysis. Selected percentiles of body weight, height/length, and body mass index were calculated according to sex, and growth curves were drawn using a spline function. The size deficit of the twins compared to the standards for the general population of Japan was calculated. RESULTS: Gestational age, parity, zygosity, and birth order affected physical growth in varying degrees, although the overall effects themselves were small and mostly disappeared by one year of age. Growth charts of the twins present growth at selected percentiles from birth to 6 years of age according to sex. The size deficit of the twins was largest at birth: more than 20% for weight and approximately 6% for length compared to the 50th percentile of the standard for the general population of Japan. These deficits decreased rapidly in the first 6 to 12 months, and were found to be as low as 0-2% at 4 to 6 years of age. CONCLUSION: Growth charts specifically for twins are needed, at least for the first 1 to 3 years of age but not beyond the age of 6 years.

Pronuclear scoring. Time for international standardization.

Zygote scoring is an efficient tool for embryo selection not only in countries where embryo selection is not permitted. Several different scoring systems have been published so far, making comparisons of assessments between investigators and laboratories extremely difficult. Pronuclear evaluation should be standardized in a manner analogous to the standardization of cleavage stage embryo scoring or of semen evaluation by the World Health Organization. The ideal score should be clear and easily applicable. The items that have the greatest influence on embryonic development seem to be alignment and size of pronuclei, alignment and number of nucleoli, halo effect and appearance of vacuoles. These morphologic parameters can be observed in different features and can be summarized as a zygote score.

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes.

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19-25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART.

Constant multiple birth rates in the Czech Republic and the Slovak Republic until recently, 1972-1995.

Using vital statistics, yearly changes in the twinning and triplet rates by zygosity were investigated in the Czech Republic and the Slovak Republic during the period 1972-1995. Monozygotic (MZ) twinning rates in both countries had remained nearly constant (about 3 per 1000 total births) during that period. With a few exceptions, the dizygotic (DZ) twinning rates remained constant from 1972 to 1994, and increased in 1995 for both countries. MZ twinning rates for both countries were the lowest in Europe. As for triplet rates, overall rates increased significantly year by year in the Czech Republic, but not in the Slovak Republic. The triplet rate was significantly higher in 1995 than in the period 1972-1982 for both countries. The MZ triplet rate remained constant during that period in the Czech Republic. The trizygotic (TZ) triplet rates increased 3-fold for the Czech Republic and 4-fold for the Slovak Republic in 1972-1976 and 1992-1995. In the later period, the TZ rate was 1.5-fold higher in the Czech Republic than in the Slovak Republic. The quadruplet rate increased 2.3-fold from 2.9 per million births in 1982-1986 to 6.7 in 1992-1995 in the Czech Republic. The corresponding values were 2.7, 2.20 and 5.9-fold in the Slovak Republic. Both the Czech and the Slovak Republics were not affected by fertility drugs and assisted reproductive techniques until recently.