DOCSLIB.ORG

Cinematographic Analysis of Bovine Embryo Development in Serum-Free Oviduct-Conditioned Medium B

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cinematographic Analysis of Bovine Embryo Development in Serum-Free Oviduct-Conditioned Medium B Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium B. Grisart, A. Massip and F. Dessy Université Catholique de Louvain, Unité des Sciences Vétérinaires, Louvain-la-Neuve, Belgium Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three\p=n-\four-cell,five\p=n-\eight-cell,9\p=n-\16-cell,morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11\p=n-\62h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula\p=n-\blastocysts(3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three\p=n-\four-celland five\p=n-\eight-cell),the higher the probability that they developed into morula\p=n-\blastocyst:70% of the embryos reaching the two-cell stage before 30\p=n-\31h after insemination developed into morula\p=n-\blastocyst.We suggest that the lag-phase as well as the link between early cleavage kinetics and further development could be related to the transcriptional activity of the embryo at about the 5\p=n-\8-cellstage. Introduction 1991; Van Soom et ah, 1992) because these investigations require frequent observations out of the incubator and could In vitro methodology to obtain large numbers of bovine affect normal embryo development. The only way to obtain embryos is routinely used, and even on an industrial scale to accurate data is by continuous cinematographic recording of produce beef embryos of desired genetic quality (Gordon, embryos in culture. This method has been used previously to 1991; Lu and Polge, 1992). This method is also an excellent observe cow embryos harvested in vivo (Massip and Mulnard, tool for fundamental investigations of early embryo develop¬ 1980; Massip et ah, 1982, 1983a, b). ment. In this respect much work remains to be done in In the present study time-lapse cinematography was used to livestock species such as cattle. At least three critical stages in analyse the behaviour of embryos cultured in serum-free embryo development can be influenced by culture conditions: oviduct-conditioned medium. The aim was to obtain infor¬ (1) the transition from maternal to zygotic control of develop¬ mation on the developmental characteristics of individual ment; (2) compaction at the morula stage; and (3) blastocyst embryos and thus to establish a correlation between the early formation. Serum-free oviduct-conditioned media support behaviour of an embryo and its further development in vitro. development from the one-cell to the blastocyst stage (Mermillod et ah, 1992a, b, 1993). An accurate study of the timing of embryo development in Materials and Methods this kind of media would provide interesting information on preimplantation features, such as cell-cycle duration and the Source of embryos and culture lengthening of the cell cycle at the time when genomic Embryos were produced by in vitro maturation and in vitro expression resumes. fertilization of oocytes from ovaries of cows, Few have been on kinetics of slaughtered reports published cleavage to the method described Mermillod et ah bovine in vitro (Sirard and Lambert, 1985; according by (1992b). embryos produced Intact were matured in tissue First and Barnes, 1989; Pollard et ah, 1991; Barnes and First, cumulus-oocyte complexes culture medium 199 plus 10% heat-treated fetal calf serum with 0.5 FSH ml , 5 *Correspondence: Unité Vétérinaire, Place Croix du Sud 3, B-1348 Louvain- supplemented µg pure pig ~ µg pure pig la-Neuve, Belgium. LH ml-1 (Beckers, 1987) and 1 µg oestradiol ml"1 (Sigma Received 6 July 1993. Chemical Company, St Louis, MO). One hundred complexes Downloaded from Bioscientifica.com at 10/04/2021 06:03:28AM via free access were placed in 500 µ of this medium in four-well plates (Nunc, day culture began was day 0. On day 2, rates of cleavage and Roskilde). After 24 h at 39°C in a humidified atmosphere of formation of five—eight-cell embryos were recorded for both containing 5% C02, cumulus-oocyte complexes with groups. The rates of formation of blastocysts on days 6, 7 and expanded cumulus masses were transferred to fertilization 8 were also recorded for both groups. medium in four-well plates. Three movies were performed, one at 10 magnification This medium consisted of modified Tyrode's medium (movie 416) and two at 5 magnification (movies 477 and with 6 BSA ml , was used more supplemented mg fatty-acid-free ~ 4 mg 483). The higher magnification to obtain sodium lactate ml-1, 0.11 mg sodium ml-1 accurate data on each (for rate and pyruvate x embryo example cleavage Chemical and 10 ml exact The same conditioned medium (Sigma Company) (TALP) µg heparin ~ number of cells). batch of (Calbiochem, San Diego, CA). To each 500 µ of fertilization was used throughout. medium 2 x 10 spermatozoa, separated using Percoli (Pharmacia, were added. The same ejaculate from one Uppsala), Statistical Belgian Blue bull was used throughout the experiments. analysis were removed from the Cumulus-oocyte complexes fertiliz¬ Chi-square analysis (P < 0.05) was used to compare the ation medium after 18 and were denuded from h, oocytes number of embryos at different stages. ANOVA 1 was used to cumulus cells at medium min. About by vortexing speed for 2 compare mean cleavage time. 100 ova were handled together in 2 ml PBS containing 0.25% trypsin (Gibco BRL, Paisley). Only zygotes that were com¬ pletely cumulus-free were placed in culture. This was to avoid Results possible depletion of the culture medium by granulosa cells. After washing in medium 199, naked zygotes were cultured Validity of the time-lapse analysis method at 39°C in droplets (50 embryos per 50 µ ) of serum-free oviduct-conditioned medium (Mermillod et ah, 1992a, 1993) A total of 250 zygotes were cultured, 111 in the incubator and 139 in the chamber. 130 under mineral oil in a humidified atmosphere containing 5% cinematographic However, only of the in the chamber were because some C02. The culture was run for 8 days in the same drop of zygotes analysed conditioned medium without replenishing the media. embryos shifted out of the field of the cine-camera in movie 416. The number of embryos reaching the two—four-cell and five-eight-cell stage after 2 days of culture, and the cumulative Time-lapse cinematographic equipment number of blastocysts on days 6, 7 and 8 were recorded in both groups (Table 1). A box cm cm Plexiglas thermoregulated (60 35 50 cm) On day 2, 37-74% of embryos were at the five—eight cell was adapted to fit onto an inverted microscope (Nikon stage in the chamber, with 41—55% thermometer located cinematographic compared Diaphot, Tokyo). A contact close to the in the incubator. The proportion of blastocysts at day 8 varied a domestic and cinematographic chamber controlled hair-dryer from 15 to 21% under the camera versus 17 to 24% in the maintained an internal of 39°C. A temperature cinematographic incubator. No significant difference (P > 0.05) between control chamber on the was built first placed microscope plate by and experimental groups was observed at any stage of the boring a hole, 6.5 cm in diameter, into a Plexiglas sheet 2 cm three movies. The rates of formation of five—eight-cell stage thick. A bottom sheet 2 mm thick was into a sheet glued place; embryos on day 2 and of blastocysts on days 6, 7 and 8 did not 6 mm thick was screwed with thumb screws onto the and top; differ significantly from one movie to another in the control the entire structure secured a were cultured by gasket. Embryos group (I) or in the experimental (C) group. in a tissue culture dish 3.5 cm in diameter (Falcon 3080, Becton Dickinson, Erembodegem) placed directly inside the closed cinematographic chamber. A 5% C02:95% air mixture was Kinetics of development humidified and warmed by it sterile water at passing through Distribution of cleavage divisions. The distribution of 39°C in a gas bubbler bottle and flushed into the chamber for embryos at each stage the culture 15 min every 30 min. The culture was run for 8—10 developmental during period days is shown The distribution curves and the without changing the medium. (Fig. 1). overlap progressive decrease of the height of the for the succes¬ A Bolex 16 mm cine-camera controlled by a peak time-lapse sive stages indicates the progressive block of embryo develop¬ system exposed a single frame every minute. The microscope ment. The curve for the five-eight-cell stage (Fig. lc) is light was switched on a few seconds before and switched off characterized by a plateau and a slow decline. It is followed at immediately after.
Load more

Recommended publications
  • 3 Embryology and Development
    BIOL 6505 − INTRODUCTION TO FETAL MEDICINE 3. EMBRYOLOGY AND DEVELOPMENT Arlet G. Kurkchubasche, M.D. INTRODUCTION Embryology – the field of study that pertains to the developing organism/human Basic embryology –usually taught in the chronologic sequence of events. These events are the basis for understanding the congenital anomalies that we encounter in the fetus, and help explain the relationships to other organ system concerns. Below is a synopsis of some of the critical steps in embryogenesis from the anatomic rather than molecular basis. These concepts will be more intuitive and evident in conjunction with diagrams and animated sequences. This text is a synopsis of material provided in Langman’s Medical Embryology, 9th ed. First week – ovulation to fertilization to implantation Fertilization restores 1) the diploid number of chromosomes, 2) determines the chromosomal sex and 3) initiates cleavage. Cleavage of the fertilized ovum results in mitotic divisions generating blastomeres that form a 16-cell morula. The dense morula develops a central cavity and now forms the blastocyst, which restructures into 2 components. The inner cell mass forms the embryoblast and outer cell mass the trophoblast. Consequences for fetal management: Variances in cleavage, i.e. splitting of the zygote at various stages/locations - leads to monozygotic twinning with various relationships of the fetal membranes. Cleavage at later weeks will lead to conjoined twinning. Second week: the week of twos – marked by bilaminar germ disc formation. Commences with blastocyst partially embedded in endometrial stroma Trophoblast forms – 1) cytotrophoblast – mitotic cells that coalesce to form 2) syncytiotrophoblast – erodes into maternal tissues, forms lacunae which are critical to development of the uteroplacental circulation.
    [Show full text]
  • Developmental Rate and Behavior of Early Life Stages of Bighead Carp and Silver Carp
    Developmental Rate and Behavior of Early Life Stages of Bighead Carp and Silver Carp Scientific Investigations Report 2011–5076 U.S. Department of the Interior U.S. Geological Survey Cover images. Left to right (top) silver carp embryo at morula stage, silver carp embryo at olfactory placode stage, silver carp embryo at otolith appearance stage, (bottom) silver carp larvae at gill filament stage, silver carp larvae at one-chamber gas bladder stage. Developmental Rate and Behavior of Early Life Stages of Bighead Carp and Silver Carp By Duane C. Chapman and Amy E. George Scientific Investigations Report 2011–5076 U.S. Department of the Interior U.S. Geological Survey U.S. Department of the Interior KEN SALAZAR, Secretary U.S. Geological Survey Marcia K. McNutt, Director U.S. Geological Survey, Reston, Virginia: 2011 For more information on the USGS—the Federal source for science about the Earth, its natural and living resources, natural hazards, and the environment, visit http://www.usgs.gov or call 1–888–ASK–USGS. For an overview of USGS information products, including maps, imagery, and publications, visit http://www.usgs.gov/pubprod To order this and other USGS information products, visit http://store.usgs.gov Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Although this report is in the public domain, permission must be secured from the individual copyright owners to reproduce any copyrighted materials contained within this report. Suggested citation: Chapman, D.C., George, A.E., 2011, Developmental rate and behavior of early life stages of bighead carp and silver carp: U.S.
    [Show full text]
  • From Cell Death to Embryo Arrest: Mathematical Models of Human Preimplantation Embryo Development
    From cell death to embryo arrest: Mathematical models of human preimplantation embryo development K. Hardy*†‡, S. Spanos*, D. Becker†§, P. Iannelli†¶, R. M. L. Winston*, and J. Stark†¶ *Department of Reproductive Science and Medicine, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom; §Department of Anatomy and Developmental Biology, ¶Centre for Nonlinear Dynamics and Its Applications, and †CoMPLEX (Centre for Mathematics and Physics in the Life Sciences and Experimental Biology), University College London, Gower Street, London WC1E 6BT, United Kingdom Communicated by Paul Nurse, Imperial Cancer Research Fund, London, United Kingdom, October 18, 2000 (received for review March 23, 2000) Human preimplantation embryos exhibit high levels of apoptotic generations during which individual cells can divide or die. The cells and high rates of developmental arrest during the first week relationship between the rate of cell death for individual cells in vitro. The relation between the two is unclear and difficult to and the data in Fig. 2 is therefore complex, precluding the direct determine by conventional experimental approaches, partly be- use of standard statistical tests. Instead, we construct a mathe- cause of limited numbers of embryos. We apply a mixture of matical model of the cell division and cell death process that experiment and mathematical modeling to show that observed allows us to relate parameters such as individual cell death rates levels of cell death can be reconciled with the high levels of embryo to global outcomes, such as the distribution of live and dead cell arrest seen in the human only if the developmental competence of numbers or the arrest or survival of the whole embryo.
    [Show full text]
  • Development Development
    DEVELOPMENT DEVELOPMENT • Prenatal –Before birth • Postnatal development- After birth • PRENATAL DEVELOPMENT – 1. Embryonic development – Up to 8 weeks after fertilization. Devided into 23 arbitrarory stages called as Carnegie Stages Pre implantation development Post implantation development 2.Foetal development 8 weeks onwards after fertilization Cleavage ( post fertilization) • process of subdivision of ovum into smaller cells called cleavage. • process of repeated mitotic divisions of zygote occur with in zonapellucida, • these cells are known as blastomeres, • first cleavage division occur around 24 hrs after fertilization, • during 8 cell stage compaction of cells occur within the cells flatten & increase their intercellular contact , • Cleavage proceed to 16 celled stage --- MORULA, • All cells of approximately same size, • At 16 cells stage cells polarity is determuned to form outer trophoectoderm & inner cell mass, • inner cell mass give rise to embryo in future, while outer cell mass is destined to form the fetal membranes including placenta • the inner cell mass also called embryoblast , • cells of trophoblast help to provide nutrition to embryo, blastocyst • some fluid now passes into morula from uterine cavity , & partially separate the cells of inner cell mass from trophoblast. • as quantity of fluid increases the morula acquires shape of a cyst,the cells of trophoblast flattens out & inner cell mass gets attach to one side only, • the morula now is called blastocyst ,cavity is called blastocoele. • site where blastocyst is attach to inner cell mass is calld embryonic or animal pole , while opposite site is aembryonic pole. Zona pellucida( function) • trophoblast has property of being able to stick to uterine ( or other) epithelium & its cells have capacity to eat up other cells( property of invading) • thus as embryo is travelling down the uterine tube & uppermost part of uterine cavity , it is prevented from sticking to epithelium by a zona pellucida.
    [Show full text]
  • Early Embryonic Development Till Gastrulation (Humans)
    Gargi College Subject: Comparative Anatomy and Developmental Biology Class: Life Sciences 2 SEM Teacher: Dr Swati Bajaj Date: 17/3/2020 Time: 2:00 pm to 3:00 pm EARLY EMBRYONIC DEVELOPMENT TILL GASTRULATION (HUMANS) CLEAVAGE: Cleavage in mammalian eggs are among the slowest in the animal kingdom, taking place some 12-24 hours apart. The first cleavage occurs along the journey of the embryo from oviduct toward the uterus. Several features distinguish mammalian cleavage: 1. Rotational cleavage: the first cleavage is normal meridional division; however, in the second cleavage, one of the two blastomeres divides meridionally and the other divides equatorially. 2. Mammalian blastomeres do not all divide at the same time. Thus the embryo frequently contains odd numbers of cells. 3. The mammalian genome is activated during early cleavage and zygotically transcribed proteins are necessary for cleavage and development. (In humans, the zygotic genes are activated around 8 cell stage) 4. Compaction: Until the eight-cell stage, they form a loosely arranged clump. Following the third cleavage, cell adhesion proteins such as E-cadherin become expressed, and the blastomeres huddle together and form a compact ball of cells. Blatocyst: The descendents of the large group of external cells of Morula become trophoblast (trophoblast produce no embryonic structure but rather form tissues of chorion, extraembryonic membrane and portion of placenta) whereas the small group internal cells give rise to Inner Cell mass (ICM), (which will give rise to embryo proper). During the process of cavitation, the trophoblast cells secrete fluid into the Morula to create blastocoel. As the blastocoel expands, the inner cell mass become positioned on one side of the ring of trophoblast cells, resulting in the distinctive mammalian blastocyst.
    [Show full text]
  • New Insights Into Human Early Embryo Development: a Particular Theoretical Study
    14 International Journal of Modern Anthropology Int. J. Mod. Anthrop. (2018) Vol: 2, Issue No: 11, pp: 14 - 46 Available online at: www.ata.org.tn ; DOI: http://dx.doi.org/10.4314/ijma.v2i11.1 Original Synthesis Article New insights into human early embryo development: a particular theoretical study Hassen Chaabani Laboratory of Human Genetics and Anthropology, Faculty of Pharmacy, University of Monastir, 5000 Monastir, Tunisia. E.mail: [email protected] (Received 18 January 2018; Accepted 5 Mars 2018; Published 5 May 2018) Abstract – The experimental research on the subject of human early embryo development has been remained insufficient because of ethical norms and legal constraints. In this context, the carrying out of theoretical studies could speed up the slow knowledge progression on this subject. Thus, I present here a particular model of theoretical study based on a synthesis of selected published experimental results combined with what has been provided from my interpretation of scientific signs masked in some Qur‟an verses. In the obtained detailed scenario, I consider that the third cleavage, resulting in 8 blastomeres, is coupled to a particular rearrangement in which 2 daughter cells seem move down and 2 others move up; while the 4 remaining cells seem stay at their initial position (I called them “4 HI cells”). Nevertheless, I consider that the fourth cleavage, resulting in 16 blastomeres, is coupled to a rearrangement that gives the impression of a harmonious descent: in fact, one of each two daughter cells seems pushed down. This descent materializes a morula top–bottom axis, which will go to coincide with the embryo-abembryo axis of the blastocyst.
    [Show full text]
  • Fresh Blastocyst Embryo Transfer Is Superior to Morula Embryo Transfer in Young Patients Undergoing in Vitro Fertilization
    Open Access Austin Journal of Reproductive Medicine & Infertility Research Article Fresh Blastocyst Embryo Transfer is Superior to Morula Embryo Transfer in Young Patients Undergoing in Vitro Fertilization Malik S1, Balassiano E1, Hobeika E1, Knochenhauer ES1,2 and Traub ML1,2* Abstract 1 Department of Obstetrics and Gynecology, Staten Island Objective: To determine if blastocyst embryo transfer yields better University Hospital, USA, 475 Seaview Avenue, Staten pregnancy outcomes compared to morula embryo transfer for fresh and frozen Island, NY, USA cycles and in donor oocyte recipients. 2Island Reproductive Services, USA, 1110 South Avenue, Suite 305, Staten Island, NY, USA Study Design: Retrospective cohort of patients undergoing in vitro fertilization at a single center. Fresh, frozen, and donor egg recipient cycles *Corresponding author: Traub ML, Department of between January 1, 2008 and December 31, 2012 were studied. Patients were Obstetrics and Gynecology, Staten Island University excluded if they were considered poor prognosis and underwent day 3 embryo Hospital, Island Reproductive Services, USA transfers. Received: April 16, 2015; Accepted: June 29, 2015; Results: In patients under age 35 undergoing fresh IVF cycle, implantation Published: June 30, 2015 rates (52% v 29%, p<0.01), clinical pregnancy rates (63% v 38%, p=0.001), and live birth rates (54% v 33%, p<0.01) were all higher after blastocyst embryo transfer. No differences were seen in other SART age groups during fresh IVF. For patients undergoing FET and in donor oocyte recipients, no differences in any pregnancy outcome were between blastocyst and morula embryo transfer. Conclusions: Blastocyst embryo transfer was found to improve pregnancy outcomes in young patients undergoing fresh IVF.
    [Show full text]
  • Improves Development of One-Cell Random-Bred Mouse Embryos in Vitro J
    The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro J. J. G. Brown and D. G. Whittingham Medical Research Council, Experimental Embryology and Teratology Unit, St George's Hospital Medical School, London SW17 ORE, UK Summary. Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation develop- ment of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyru- vate and glucose, lactate pyruvate and glucose for the first 24 h, and medium contain- ing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Sub- sequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development. Keywords: preimplantation; embryo; blastocyst; two-cell block; culture; mouse Introduction Mouse embryos of all strains can develop into blastocysts when cultured from the late two-cell stage in simple chemically defined physiological salt solutions containing the energy substrates lactate, pyruvate and glucose (Brinster, 1965a, b; Biggers et al., 1967, 1971).
    [Show full text]
  • Role of Gonadotropin-Releasing Hormone (Gnrh) During Early Embryonic Development UCARE Amy R
    Role of Gonadotropin-Releasing Hormone (GnRH) During Early Embryonic Development UCARE Amy R. Cropp, Marcelo M. Montagner, Rebecca A. Cederberg, Jessica J. Swanson, and Brett R. White Undergraduate Creative Activities & Research Experiences IANR Department of Animal Science, University of Nebraska-Lincoln Materials and Methods Abstract Superovulation With human infertility rates on the rise, improved culture of oocytes and embryos can greatly enhance in At least three mature female mice were used per experiment. vitro maturation (IVM) and in vitro fertilization (IVF) techniques. Commonly, GnRH is used to enhance culture Results Mature female mice were superovulated with I.P. injections of 5 IU PMSG (Day 1) and 5 IU hCG approximately 48 methods utilized by human IVF clinics. Historically, the primary function of GnRH is stimulation of luteinizing hours later (Day 3). hormone (LH) and follicle stimulating hormone (FSH) release from the pituitary gland. In contrast, there is also evidence that GnRH might play an important role in other reproductive organs. In previous in vitro studies, Following the hCG injection, each female mouse was mated with a fertile male. 80 GnRH analogs have been associated with improved oocyte maturation, fertilization, and embryo development 100 90 The following morning (Day 4) the females were checked for cervical plugs, which indicates breeding has taken place. rates in the cow, mouse, and human (1-4). To examine the role of GnRH on mouse embryo development, we 70 80 cultured preimplantation embryos in the presents of a specific antagonist of GnRH (SB-75), a specific GnRH 60 Media 70 Morula 50 agonist (Histrelin), and a combination of treatments.
    [Show full text]
  • Label-Free Assessment of Pre-Implantation Embryo
    www.nature.com/scientificreports OPEN Label-free assessment of pre- implantation embryo quality by the Fluorescence Lifetime Imaging Received: 10 July 2018 Accepted: 28 June 2019 Microscopy (FLIM)-phasor approach Published: xx xx xxxx Ning Ma1,2, Nabora Reyes de Mochel3,4, Paula Duyen Pham3, Tae Yeon Yoo3, Ken W. Y. Cho3 & Michelle A. Digman1,2,3 Development of quantitative, safe and rapid techniques for assessing embryo quality provides signifcant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fuorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non- hypoxic conditions, the unique spectroscopic trajectories at diferent stages of mouse pre-implantation development, which is referred to as the developmental, or “D-trajectory”, that consists of fuorescence lifetime from diferent stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fuorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defned a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fuorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non- invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.
    [Show full text]
  • The First 21 Days
    The First 21 Days k and myst Ris ery surround e embryo’s ear th liest development ne of life’s most profound mysteries — the creation of a new human being — starts with the union of egg and sperm and ends, nine months later, with a baby. It’s a process so intricate and inherently risky, with pitfalls at every stage, it’s amazing any of us survive long enough to be born at all. In fact, scientists estimate that about half of all human embryos die during the first two months of pregnancy when something goes wrong in those critical early stages of development. It takes two months to form the human embryo, a process scientists call embryogenesis. After eight weeks, the embryo has all the organs and tissues found in a newborn baby — although many exist in primitive form. During the last seven months of a human pregnancy, the fetus (no longer called an embryo) continues to grow and develop, but the fundamental blueprint for the baby-to-be is Oestablished during embryogenesis. 22 BY SALLY POBOJEWSKI NIKAS YORGOS MEDICINE AT MICHIGAN O A human egg surrounded by sperm (color-enhanced) PHOTO CREDIT PHOTO 23 WWW.MEDICINEATMICHIGAN.ORG/MAGAZINE Many of the most important and least understood stages in-vitro fertilization (IVF) or other assisted reproductive of a human embryo’s development take place during the first technologies. In 2004, 49,458 babies were born in the U.S. 21 days of pregnancy before the mother even knows she’s from embryos created when sperm and egg got together in pregnant and while the embryo is still incredibly small.
    [Show full text]
  • General Embryology-2-Early Development
    Fertilization Process of fusion of male and female gametes in the ampullary region of the fallopian tube. • Entrance of sperms in the female genital tract. • Capacitation • Acrosome reaction •Penetration of corona radiata •Penetration of zona pellucida •Fusion of oocyte and sperm cell membrane Response of ovum •Cortical and zona reactions. •Resumption of Meiosis II •Metabolic activation of the egg. Results of fertilization •Determination of sex •Restoration of diploid number of chromosomes •Initiation of cleavage Zygote is -2 celled at 30 hrs -4 celled at 40 hours -12 celled at 3 days -16 celled at 4 days •This 16 celled stage is –morula •Compaction: morula cells form an inner and outer cell mass •zona pellucida disappears at 4th day 30 8 69 5th day 6th day SUMARY OF DEVELOPMENT IN 1st WEEK AFTER FERILIZATION Cleavage Morula Blastocyst formation Implantation 8th day 9th day 12th day 13th day Connecting stalk SUMARY OF DEVELOPMENT IN 2ND WEEK Completion of embedding. TROPHOBLASTS EMBRYOBLASTS • Differentiate into • Differentiate into cytotrophoblast & epiblast & hypoblast syncytiotrophoblast • Formation of amnioblasts and • Lacunae formed & amniotic cavity beginning of • Formation of primary and uteroplacental circulation secondary yolk sacs • Formation of primary villi • Formation of extraembryonic Decidual Reaction: mesoderm; ee coelom (chorionic cavity); -Oedema of stroma ee somatopleuric and -Enlarged glands splanchnopleuric layers -Increased Glycogen content of epithelial cells -Congestion ECTOPIC IMPLANTATION • Tubal • Ampullary • Interstitial • Low uterine •Ovarian • Secondary abdominal NOTOCHORD • Provides midline axis to the body. • Induces formation of neural plate. REMAINS OF NOTOCHORD 1. Nucleus pulposus. 2. Apical ligament. 3. Its cranial part is incorporated into basiocciput and basisphenoid. Approx. Age (Days) No.
    [Show full text]