Classification Of Embryos Research Articles

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P > 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P < 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P > 0.05). However, blastocyst rate in G2 (25.0%) was higher (P < 0.05) than in G1 and G3, but was similar to G4 (P > 0.05). Total cell number was similar (P > 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P < 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P < 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P > 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

Changes in biomass and elemental composition during early ontogeny of the Antarctic isopod crustacean Ceratoserolis trilobitoides

Changes in biomass and elemental composition (dry mass, DM; carbon, C; hydrogen, H; nitrogen, N) were studied throughout the early ontogeny in the serolid isopod Ceratoserolis trilobitoides from a population off the South Shetland Islands (62°24.35′S, 61°23.77′W). Specimens of C. trilobitoides were sampled using an Agassiz trawl during the expedition ANT XXIII-8 of RV Polarstern in January 2007. Classification of embryos into six developmental stages followed previous studies. No clear size-dependant fecundity relationship was found in ovigerous C. trilobitoides. Egg volume increased by about 160 and 400% from stage I to IV and stage IV to VI, respectively. DM, C, N, and H continuously decreased throughout the early ontogeny from stage I to VI, but DM showed significant increase on reaching the late-V stage and premanca stages. The C:N ratio remained relatively stable throughout stages I to V, followed by a significant drop from about 6.17 to 5.5 in subsequent stages, indicating depletion of lipid resources of maternal origin. The results coincide with previous studies and indicate a shift from a lipid-based metabolism throughout early embryo stages to a protein-based metabolism in the late-V and premanca stage, which requires external energy supply. Given the steep increase in DM in the final phase of embryo development (late-V stage to premanca) and the need for external food supply to exert growth, the possibility of external food supply or cannibalism in early offspring of C. trilobitoides is discussed.

Sperm retention site and its influence on pronucleus stage evaluation following intracytoplasmic sperm injection

Aim: It has been suggested that the position of the sperm after intracytoplasmic sperm injection (ICSI) has an effect on the development and quality of the embryo. In this study, we retrospectively examined whether pronucleus stage evaluation used through clinical studies in recent years has relevance with regard to sperm location. Methods: From 2003 to 2005, 1285 oocytes from 459 patients (average age: 36years) were retrospectively analyzed. The 459 patients underwent ICSI because of fertilization disorders and oligozoospermia. Follicle stimulation was via either Clomid or the long protocol. Human chorionic gonadotropin was administered to induce ovulation and oocyte retrieval was conducted 35h later. After confirming the presence of a polar body, we immobilized the ovum at the 6 o'clock position, introduced the injection pipette at the 3 o'clock position and carried out ICSI. Results: When a sperm was located at a position that was opposite to the polar body, both classifications of Scott and Tesarik regarding embryo quality were distinctly low. Furthermore, a good embryo classification ensued when the sperm was located adjacent to the polar body. Conclusion: The zone in which the sperm was located did not always correlate with embryo quality; however, our study suggested that sperm location affects the synchronization of the nucleolus. When carrying out ICSI, it is important to take into consideration the insertion point of the sperm. (Reprod Med Biol 2007; 6: 171-174).

P-5: Cumulus cell apoptosis quantification: Possible clinical implications in IVF programs

Degree of granulosa/cumulus cell apoptosis is a hallmark of antral follicular atresia, may contribute to reduced ovarian reserve and, may be responsive to exogenous gonadotropins in controlled ovarian stimulation (COS). Currently it is controversial as to whether all follicles under COS have similar degrees of granulosa/cumulus cell apoptosis, and whether the degree of apoptosis might be predictive of oocyte and embryo quality and successful pregnancy. Granulosa/cumulus cell apoptosis has been quantified using fluorescent microscopy and manual cell counting, however such methods are time-consuming and laborious. Conversely, flow cytometry may be advantageous in its ability to measure quantitatively large numbers cells in a short time. This study aimed to investigate a simple, quantitative method using fluorescent flow cytometry to compare cumulus cell apoptosis in patients with infertility, undergoing COS and in vitro fertilization (IVF). Prospective study. Seventeen patients 26 to 44 years of age (mean ± SD: 35.3 ± 5.1) were included in this study. After ovarian stimulation, cumulus complexes (CCs) were mechanically isolated from 101 oocytes obtained from follicles ≥ 10mm in diameter. Mature oocytes were inseminated by ICSI with motile sperm. Embryo culture was performed in individual 50μl drops and embryo development was assessed by morphological observations and graduated embryo scoring was performed every 24h until embryo transfer on day 3. Cumulus cells were treated with 80IU hyaluronidase followed by triplicate washes in PBS to obtain a cumulus single-cell suspension. Cumulus cell apoptosis was measured using fluorescent flow cytometry (Guava PCA, USA) and DNA histograms were analyzed using the CytoSoft software (version 2.1.4). Kruskal-Wallis Test, one-way ANOVA and t-test were used as appropriate. Differences were considered significant at P<0.05. On average cumulus complexes contained 49,992±20,719 cells (Mean number ± SD). Of these, 27.5% were classified as viable, 8.0% as mid-apoptotic cells and 64.5% dead. There was no relationship between maternal age, type of CC, number of retrieved oocytes and embryo classification and percentage of viable, mid-apoptotic or dead cells. Main outcome measures are shown in table I.Tabled 1 Patients undergoing IVF with serum FSH > 5mIU/mL have preovulatory follicles with an increase in percentage of cells undergoing apoptosis as compared with women with serum FSH ≤ 5mIU/mL. The preliminary results suggested that oocyte developmental competence was not associated with CC apoptosis.

Interobserver agreement and intraobserver reproducibility of embryo quality assessments

The objective of this investigation was to determine the inter- and intraobserver agreement when assessing embryo quality. This investigation included 4002 cleaved embryos from 7535 oocytes retrieved in 688 patients undergoing IVF cycles in a multicentre trial. Embryos were evaluated locally at the inverted microscope at 28, 44 and 68 h (+/-1 h) post-insemination. Digital images of the embryos were assessed centrally by three blinded embryologists. To assess reproducibility, 215 randomly selected cleaved embryos from 33 patients were re-evaluated by the three central embryologists. The interobserver agreement among the central embryologists (using the same method of evaluation; 2D images) was good for classification of top-quality embryos (kappa 0.71-0.73), excellent for classification of normally developed embryos (kappa 0.83-0.86) and good-excellent for classification of transferable embryos (kappa 0.78-0.82). The interobserver agreement between local and consolidated central assessment (different methods of evaluation, inverted microscopy versus 2D images) was good for all three embryo classifications (kappa 0.64-0.79). The intraobserver reproducibility for all three overall embryo classifications was excellent for the consolidated central assessment (kappa 0.80-0.91). Embryo quality can be determined with a good degree of interobserver agreement independently of the method of evaluation. Embryologists classify embryos with excellent intraobserver reproducibility.

Mosaic and regulative development: two faces of one coin

“There seems to be no more a completely mosaic egg than a completely regulation one” Waddington, Principles of Embryology 1956, Allen and Unwin, p63.

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 +/- 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 +/- 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

Interobserver Agreement and Intraobserver Reproducibility of Embryo Quality Assessments

Background The objective of this investigation was to determine the inter- and intraobserver agreement when assessing embryo quality. Methods This investigation included 4002 cleaved embryos from 7535 oocytes retrieved in 688 patients undergoing IVF cycles in a multicentre trial. Embryos were evaluated locally at the inverted microscope at 28, 44 and 68 h (+/-1 h) post-insemination. Digital images of the embryos were assessed centrally by three blinded embryologists. To assess reproducibility, 215 randomly selected cleaved embryos from 33 patients were re-evaluated by the three central embryologists. Results The interobserver agreement among the central embryologists (using the same method of evaluation; 2D images) was good for classification of top-quality embryos (kappa 0.71-0.73), excellent for classification of normally developed embryos (kappa 0.83-0.86) and good-excellent for classification of transferable embryos (kappa 0.78-0.82). The interobserver agreement between local and consolidated central assessment (different methods of evaluation, inverted microscopy versus 2D images) was good for all three embryo classifications (kappa 0.64-0.79). The intraobserver reproducibility for all three overall embryo classifications was excellent for the consolidated central assessment (kappa 0.80-0.91). Conclusion Embryo quality can be determined with a good degree of interobserver agreement independently of the method of evaluation. Embryologists classify embryos with excellent intraobserver reproducibility.

Embryology of the Mangrove Crab Ucides Cordatus (Brachyura: Ocypodidae)

Abstract Ovigerous females of Ucides cordatus were collected at different mangrove areas in Iguape, SP, Brazil, and reared in the laboratory in order to describe each embryonic stage. Accounts of the biometry, internal morphology, and coloration of the eggs were taken. The embryonic development took 19 ± 1 d (27°C, 12:12 h) with eight well-defined stages. A salinity test on embryonic development was conducted, and a better result was obtained in a rearing at 15‰. Eggs are slightly ellipsoid through development and undergo an increase of 13.9% in diameter to 91.7% in volume. Cluster analysis revealed three different groups for diameter (Stages I–II, III–VI, and VII–VIII) and volume (Stages I–III, IV–V, and VI–VIII), supporting the classification of embryos into initial, intermediate, and final stages, which are currently used in studies on the reproductive biology of decapod crustaceans. In this species, the identification of the different embryonic stages can be achieved only if biometric data are combine...

"Properly Disposed of": A history of embryo disposal and the changing claims on fetal remains

This paper explores recent controversies concerning the disposal of embryonic and fetal remains in order to ask how such remains came to be classified as "medical waste." Based on archival research into the social history of human embryo collecting in Baltimore, Maryland, in the early 20th century, I argue that the classification of embryos and fetal remains as medical waste can be traced to a pragmatic alliance between embryologists and state functionaries. Embryologists relied on the state to assist them in acquiring thousands of human embryo remains for scientific study, while state authorities relied on embryologists to provide authoritative knowledge that could be used to facilitate state control over nascent citizens. This alliance contributed to the development of an "embryological worldview," in which human embryos were cast as objective biological "specimens" of use only to embryologists. This exclusive view of the social value of embryos and fetal tissue is now being challenged as other constituencies claim jurisdiction over the remains in order to advance diverse social agendas.

Human embryo fragmentation in vitro and its implications for pregnancy and implantation

Objective: To evaluate the effects of fragmentation and fragment removal in day 3 human embryos on implantation and pregnancy. Design: Retrospective analysis of ETs homogeneous with respect to embryo fragmentation. Setting: A program of IVF-ET. Patient(s): The study population consisted of 2,410 patients. Intervention(s): The degree and pattern of fragmentation were evaluated on days 2 and 3; microsurgical fragment removal was performed after assisted hatching on day 3. Main Outcome Measure(s): Clinical pregnancy and implantation rates. Result(s): The degree and pattern of fragmentation significantly impact pregnancy and implantation. With the application of microsurgical fragment removal before ET, embryos with 6%–35% fragmentation implant with similar frequency. The presence of large fragments (type IV) is detrimental to the developing embryo, whereas localized or small and scattered fragments do not significantly affect implantation. Conclusion(s): The potential of fragmented embryos for implantation is determined partly by the distribution of fragments. Adoption of an embryo classification system reflecting types of fragmentation is advisable. The use of microsurgical fragment removal significantly alters the course of development for some embryos and improves their implanting potential.

A neural network based pattern recognition system for somatic embryos of Douglas fir

A pattern recognition system was developed to classify Douglas fir somatic embryos by employing an image analysis system and two neural network based classifiers. The contour of embryo images was segmented, digitalized and converted to numerical values after the discrete and fast Fourier transformation. These values, or Fourier features, along with some other shape factors, were used for embryo classification. The pattern recognition system used a hierarchical decision tree to classify Douglas fir embryos into three normal and one abnormal embryo classes. An accuracy of greater than 80% was achieved for normal embryos. This system provides an objective and efficient method of classifying embryos of Douglas fir. It will be a useful tool for kinetic studies and process optimization of conifer somatic embryogenesis.

Classification of Plant Somatic Embryos by Using Neural Network Classifiers

The development of somatic embryos is characterized by a series of morphological changes. Quantitative kinetic studies have been hampered by the difficulties in enumerating and characterizing embryo populations. By employing neural networks, we have developed a pattern-recognition system for characterizing the morphological features of carrot somatic embryos. This pattern-recognition system employs a hierarchical decision tree to achieve optimal classification. It successfully classified carrot somatic embryos into normal and abnormal embryo classes. For normal embryo classes (globular, oblong, heart, and torpedo embryos), an accuracy of 90% or higher was achieved. The features identified by the neural network classifiers as most important for embryo classification are almost identical to those obtained by the branch-and-bound searching algorithm used previously. However, employing the neural networks shortens the system developing time greatly. Coupled with an image analysis system, this neural-network-based pattern-recognition system shows great potential in embryo sorting and automation of synthetic seed production.

Presence of mitotic cells: A parameter of embryo quality

The aim of this study was to identify objective parameters for measuring embryo quality instead of those based on morphological appearance. We have investigated the relationship between embryo quality as assessed by morphological appearance and the number of blastomeres in bovine embryos at 5–7 days as well as the relation between embryo quality and presence of mitotic and pycnotic nuclei. Superovulated cows were slaughtered from 5 to 7 days after ovulation and embryos were flushed from the excised uteri. They were fixed in acetic acid-ethanol, and stained with orcein, following qualification according to their morphological appearance by microscopy. Blastomere numbers were determined for each day and quality. Some of the Day-6 embryos were classified according to the presence of mitotic and/or pycnotic cells. The number of blastomeres per embryo increased with increasing quality and with age except for poor embryos, where the number of blastomeres remained low. Classification of the Day-6 embryos according to the status of their cells, showed that 72% of the excellent and good embryos were categorized as having mitotic cells. Of the embryos that were classified as fair or poor, 53.5% had no visible mitotic activity. Mitotic activity is indicative of embryo quality. It is correlated with high numbers of blastomeres and might be responsible for embryo viability.

Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.

Classification of plant somatic embryos by computer vision

This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed.

The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization

To determine if a simple morphological classification of embryos was predictive of subsequent pregnancy. Prospective case series. University-based in vitro fertilization (IVF) program. Consecutive embryo transfer (ET) cycles (n = 206). Embryos were classified into three grades: (1) equal-size blastomeres with no fragmentation; (2) unequal-size blastomeres; and (3) evidence of fragmentation. Embryo quality, age, indication for IVF, and stimulation protocol were evaluated for their effect on pregnancy rates (PR's). In cycles in which the best embryo transferred was grade 3, 2, or 1, the clinical PRs per ET were 0% (0/11 cycles), 12.8% (6/47 cycles, P less than 0.05), and 21.8% (32/148 cycles, P less than 0.05), respectively. When one, two, or three or more grade 1 embryos were replaced, the clinical PRs per ET were 15.6%, 16.3%, and 40% (P less than 0.05), respectively. Using logistic regression, embryo quality (P = 0.0011) and patient's age (P = 0.0044) were the only variables that affected PRs. The transfer of more than two good quality embryos had a positive effect, patient's age had a negative effect on PRs after IVF-ET.

Treatment of the embryo and the fetus in the first trimester: Current status and future prospects

An effective treatment is now available to prevent the masculinization of female fetuses with congenital adrenal hyperplasia. Some vitamin-responsive inborn errors of metabolism can be treated prenatally by cofactor administration. Maternal phenylketonuria and maternal diabetes mellitus and the prevention of recurrent neural tube defects are also areas where therapeutic advances are being made. It may be possible to carry out chorionic villi sampling before 8 weeks menstrual age if appropriate catheters and guidance systems (probably transvaginal ultrasound) are used. First trimester diagnosis and treatment of fetal cardiac arrhythmias could prove to be very important, as they are later in pregnancy. Future possibilities for progress include gene microinjection into zygotes, classification and treatment of fresh embryos, biopsy and frozen storage of genetically at risk embryos, and therapy of preimplantation embryos by chimera formation or gene introduction by retroviruses.

Identification of embryonic sex by detection of H-Y antigen

The commercial embryo transfer industry will find immediate use for a reliable, economical and practical procedure for identification of embryonic sex prior to transfer. Unfortunately, no procedure is currently available that meets these criteria. An approach under study is detection of a male-specific factor (hereafter referred to as H-Y antigen) on early embryos. Expression of H-Y antigen by cleavage-stage mouse embryos was first reported over a decade ago, but only over the past several years has detection of H-Y antigen been considered potentially useful to the embryo transfer industry. H-Y antigen is expressed as early as the eight-cell stage and is readily detectable on morulae; detection becomes more difficult at the blastocyst stage. It has been shown to be expressed by preimplantation embryos of all mammalian species studied, including the mouse, rat, pig, sheep, goat, cow and horse. For at least four of these species assays for H-Y antigen are approximately 85% accurate in identification of embryonic sex. Indirect immunofluorescence assays for embryonic H-Y antigen are compatible with survival and continued development of the embryo, but such assays involve highly subjective evaluation and classification of fluorescent embryos. Current procedures for identification of embryonic sex by detection of H-Y antigen remain largely experimental at this time.

A Study on the Classification and Systematic Inspection of Abnormal Chick Embryos

Hitherto, the classification of the teratology of chick embryo which died during the normal incubating condition has been made on the basis of major deformities even if the abnormal structures were found in several parts of body. By a close examination of large numbers of abnormal embryos it was indicated that the deformities could not be classified into a limited number of types. The importance to inspect and describe systematically the variouse abnormalities was postulated. The items for such a systemic inspection were proposed.A card with those items for checking may enable even the not well trained examiners to inspect the teratology of many chick embryos. This inspecting system may provide the facilities for study of deformities of chick embryo in the ordinary local poultry breeding stations and contribute for the accumulation of the basic data on the occurence of abnormal development.