IVM and IVF - Prof. Luisa Gioia - a.y. 2023/2024
Reproductive Biotechnologies - 1st year
ATTENZIONE: i contenuti di questo corso sono relativi all'anno accademico 2023/2024
Indice degli argomenti
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Associate Professor in Veterinary Physiology, University of Teramo, Italy
Tutor of Master’s Degree Programme in "Reproductive Biotechnologies"
ACADEMIC QUALIFICATIONS AND ACADEMIC POSITIONS
-from 1989 to 1991, Internal student of the Biochemistry Unit, Faculty of Biological Science, University of L'Aquila;
-1991: Doctor in Biological Science cum Laude, University of L'Aquila;
-1993-1997: graduate student research of the Veterinary Physiology Unit, Faculty of Veterinary Medicine, Universities of Teramo and Bologna;
-1997: PhD in Domestic Animal Endocrinology, University of Bologna, discussing an experimental thesis entitled “Intrafollicular regulation of oocyte meiosis”;
-1997-2000: Laboratory Technician at Veterinary Physiology Unit, Faculty of Veterinary Medicine, University of Teramo;
-2000-2005: Researcher in Veterinary Physiology, Faculty of Veterinary Medicine, University of Teramo;
-from 2005: Associate Professor in Veterinary Physiology, University of Teramo, Italy;
-from 2004: Member of the Doctoral Programme in Biotechnology of Reproduction/ Molecular and Cellular Biotechnology
-from 2005: Tutor of Master’s Degree Programme in "Biotechnology of Reproduction”/"Reproductive Biotechnologies"
ACADEMIC LEADERSHIP
2012-2015: Programme Director of Master’s Degree Programme in “Biotechnology of Reproduction”
RESEARCH ACTIVITY
The over twenty years research activity, regarding basic research associated to biotechological studies applied to reproduction, has been mainly carried out on the following subjects related to mammalian species: “Biology of the oocyte”, “Biology of the sperm cell”, “Oocyte maturation in vitro”, “In vitro fertilization”, “Physiology of the Graafian follicle”, “Follicular responses to pre-ovulatory surge of gonadotrophic hormones”,
The overall research activity is documented by scientific publications and conference proceedings that can be found at the following address and link:
http://www.ncbi.nlm.nih.gov/pubmed
http://www.unite.it/UniTE/Engine/RAServePG.php/P/58511UTE0603?&VRIC_IDOC=106#A4
PURPOSE OF THE INTEGRATED COURSE AND PREREQUISITES
Purpose: The theoretical part of the course unit provides students with a comprehensive and updated information on endocrine, paracrine, cellular and molecular mechanisms involved into mammalian male and female gametes differentiation, growth, maturation and recognition/interaction. This in-depth knowledge of Gamete Biology is preliminary to critically approach the biotechnological practical activities on “In Vitro Maturation and In Vitro Fertilization” (IVM and IVF) carried out in the laboratory, that the student should be able to apply for therapeutic and research purposes at the end of the course. More detailed information can be found on the Teramo University website at the link named "Scheda insegnamento"
Prerequisites: There are no prerequisites to other course programmes. A basic knowledge on Citology, Cell Physiology, Cell Culture is considered essential and is therefore required for admission to the Degree Course. It is verified through an admission test. Before the start of the Degree Course lessons, Remedial Courses are organized to help students prepare for the test.The integrated course unit is preparatory to the course unit of “Embryology”, that is subsequent in the MS Degree Course structure diagram.
DUBLIN DESCRIPTORS FOR UNIT
UNIT1
Laboratories on In Vitro Maturation (IVM) and In Vitro Fertilization (IVF) techniques.
Setting up a laboratory: basic equipment and facilities required for a laboratory of IVM and IVF.
Oocyte IVM in domestic animals:
- Tissue culture systems used for IVM.
- Tissue culture media used for IVM.
- Selection of ovaries retrieved from slaughterhouses.
- Morphological evaluation of ovarian follicles and selection of cumulus-oocyte complexes (COCs).
- Choosing and setting up of the best culture system for oocyte IVM in domestic animals.
- Assessment of IVM oocytes.
Dublin descriptors for Unit 1
Knowledge and understanding: starting from the knowledge on Cell culture and on the detailed and updated information on endocrine, paracrine, cellular and molecular mechanisms involved in mammalian oocyte maturation, achieved through theoretical lessons, students can critically approach the practical activities on “in vitro maturation and in vitro fertilization” (IVM and IVF) techniques.
Applying knowledge and understanding: at the end of the Unit 1, first of all students shall be able to correctly manage the equipment and instrumentation of the cell culture laboratory. Then, they shall be capable of applying their knowledge to correctly set-up an IVM protocol using domestic animal oocytes. Knowledge and understanding of students, as well as their ability to apply them, will be assessed at the end of Unit1 through the formative test, and through the final examination at the end of the course unit.
Making judgements: during practical activities carried out in the laboratory, students have to demonstrate critical thinking regarding the procedures and protocols they are using. Moreover, they have to be able to critically analyze the results obtained at the end of IVM and propose a solution to any problems encountered.
Communication: the capability of students to properly communicate using a rigorous scientific terminology is continuously monitored during the practical activities carried out in the laboratory under the supervision of the teachers. That is also evaluated both during class activities and the final examination at the end of the course.
Learning skills: as many as half of the course shall focus on methodological activities carried out in specific laboratories where students may work under the teacher’s supervision, the ability of students to critically approach the practical activities on IVM techniques can be assessed in real time by teachers. That is also evaluated both during class activities and the final examination at the end of the course.
UNIT 2
IVF in domestic animals:
- Semen collection and assessment.
- Sperm preparation for IVF.
- Culture media for sperm capacitation and oocyte fertilization.
IVM and IVF procedures for in-vitro embryo production in the swine model:
- Morphological evaluation and selection of in vitro matured oocytes.
- Semen preparation for IVF.
- Protocols for sperm capacitation and IVF.
- Morphological criteria and colorimetric assay for the evaluation of fertilized oocytes.
- IVF in Humans: introduction to FIVET and in vitro early embryo development.
Dublin descriptors for Unit 2
Knowledge and understanding: making use of the theoretical background on Cell Culture and on the detailed and updated information on the biology of sperm cell, during Unit 2 students can firstly critically approach the practical activities on semen preparation and capacitation. Then, they shall be able to correctly manage an “in vitro fertilization” (IVF) protocol using IVM pig oocytes obtained according to the protocols they learned during the Unit1.
Applying knowledge and understanding: students ability to apply their knowledge to correctly handle semen samples and set-up an IVF protocol using domestic animal gametes will be assessed through a formative test at the end of the Unit 2, as well as through the final examination at the end of the course unit.
Making judgements: during the practical activities carried out in the laboratory, students have to demonstrate critical thinking regarding the procedures and protocols regarding semen preparation and IVF protocols. Moreover, they have to be able to critically analyse the results obtained at the end of IVF and propose a solution to any problems encountered.
Communication: the capability of students to properly communicate using a rigorous scientific terminology is continuosly monitored during the practical activities carried out in the laboratory under the supervision of the teachers. That is also evaluated both during class activities and the final examination at the end of the course. Students can attend seminars on Statistics organized during the MS Degree Course to improve their scientific communication skills.
Learning skills: as many as half of the course shall focus on methodological activities carried out in specific laboratories where students may work under the teacher’s supervision, the ability of students to critically approach the practical activities on IVF techniques can be assessed in real time by teachers. That is also evaluated both during class activities and the final examination at the end of the course.
UNIT 3
IVM and IVF procedures for in vitro embryo production in the ovine model
Principles and simulation of IVF techniques used in the laboratory for assisted reproduction
- Principles about human IVM and superovulation protocols.
- Culture media for oocyte retrieval (ovum pick-up), sperm capacitation and IVF.
- Morphological evaluation of COCs collected by an ovum pick-up.
- Preparation of microdroplets under mineral oil.
- Protocols for sperm capacitation and IVF in the human model.
- Morphological evaluation of zygotes obtained by IVF.
- Introduction to assessment of early in vitro embryo development
Dublin descriptors for Unit 3
Knowledge and understanding: during the last Unit, students shall be able to critically and independently approach the practical activities on IVM and IVF techniques using both swine and ovine as models. Moreover, they shall also acquire basic knowledge on the procedures and the protocols used in assisted reproduction techniques (ART).
Applying knowledge and understanding: at the end of third Unit, students shall be able to apply their knowledge to correctly handle domestic animal gametes and set-up a complete IVM/IVF protocols with the final purpose of producing embryos in vitro. The methodological skills acquired will be helpful for students to run simulations of IVF protocols commonly used in assisted reproduction techniques (ART) by using animal gametes.
Making judgements: at the end of the course, due to the theoretical educational activities in the class based on active learning method, and practical activities carried out in the laboratory side by side with the teachers, students shall develop critical thinking and acquire greater autonomy regarding the procedures and protocols used.
Communication: at the end of the course, students shall acquire the capability to properly communicate the scientific protocols and procedures learned during the course, and more generally scientific data, using a rigorous scientific terminology. They shall be also able to statistically analyze scientific data.
Learning skills: at the end of the course, students applying the detailed knowledge developed on the Biology of oocyte and sperm cell, shall acquire the methodological skills related to the management of IVM/IVF protocols using domestic animal gametes. That shall be applied by students for research purposes, and it could be also helpful for students to understand and learn IVF procedures applied on Humans for therapeutic reason.
Text books
- Kay Elder and Brian Dale, "In vitro fertilization", Cambridge University Press, Cambridge, 2011, Online ISBN:9780511984761, Paperback ISBN:9780521730723, eBook ISBN: 0511990480, Print ISBN: 0521730724, Book DOI: http://dx.doi.org/10.1017/CBO9780511984761
- Zsolt Peter Nagy, Alex C. Varghese, Ashok Agarwal, “Practical Manual of in Vitro Fertilization: Advanced Methods and Novel Devices”, Springer, New York, 2012, e-ISBN 978-1-4419-1780-5, www.springer.com
- Coticchio, Giovanni, Albertini, David F., De Santis, Lucia, "Oogenesis", Springer, New York, 2012, ISBN: 978-0-85729-825-6 (Print), e-ISBN: 978-0-85729-826-3 (Online), http://www.springer.com/medicine
- E. Knobil and J.D. Neill, “The Physiology of Reproduction", Raven Press, Ltd, New York, 2005, ISBN-10: 0125154003, ISBN-13: 978-0125154000, Versione e-book non disponibile.
Recommended textbook on CELL CULTURE
Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition
R. Ian Freshney
ISBN: 978-1-118-87365-6
March 2016, Wiley-Blackwell
In-depth material on the subjects of the course
- Jacek Z. Kubiak, Protein Kinase Assays for Measuring MPF and MAPK Activities in Mouse and Rat Oocytes and Early Embryos, Mammalian oocyte regulation: Methods and Protocols, Vol. 957, 2013, 77-89, http://link.springer.com/book/10.1007/978-1-62703-191-2/page/1
- Karl Swann, Measuring Ca2+ Oscillations in Mammalian Eggs, Mammalian oocyte regulation: Methods and Protocols, Vol. 957, 2013, 231-248, http://link.springer.com/book/10.1007/978-1-62703-191-2/page/1
- Hayden Homer, The APC/C in female mammalian meiosis I, Reproduction, Jun 27;146(2), 2013, R61-71, doi:10.1530/REP-13-0163, http://www.reproduction-online.org/content/146/2/R61.long
- Carroll J, Marangos P., The DNA damage response in mammalian oocytes, Front Genet., Jun 24;4, 2013, 117, doi: 10.3389/fgene.2013.00117, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690358
- Wakai T, Vanderheyden V, Fissore RA.Wakai T, Vanderheyden V, Fissore RA., Ca2+ signaling during mammalian fertilization: requirements, players, and adaptations. Cold Spring Harb Perspect Biol. Apr 1;3(4). 2011, pii: a006767. doi: 10.1101/cshperspect.a006767, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062211
- Sirard MA., Follicle environment and quality of in vitro matured oocytes. J Assist Reprod Genet. Jun;28(6), 2011, 483-8, doi: 10.1007/s10815-011-9554-4, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158252
- Wassarman PM, Litscher ES., Mammalian fertilization: the egg's multifunctional zona pellucida, Int J Dev Biol. 52(5-6), 2008, 665-76, doi: 10.1387/ijdb.072524pw., http://www.ijdb.ehu.es/web/paper.php?doi=10.1387/ijdb.072524pw
- Tom Ducibella, Rafael Fissore, The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development, Developmental Biology, Volume 315, Issue 2, 15, 2008, 257-79, http://www.sciencedirect.com/science/article/pii/S0012160607015989
- K. Swann, C.M. Saunders, N.T. Rogers, F.A. Lai, PLC_(zeta): A sperm protein that triggers Ca2+ oscillations
and egg activation in mammals, Seminars in Cell & Developmental Biology, 17, 2006, 264-273, www.elsevier.com/locate/sem/cdbd - Solc P., Schultz R.M. and Motlik J., Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells, Mol.Hum.Reprod., 16, 2010, 654-664, doi: 10.1093/molehr/gaq034
- Eric Rubinstein, Ahmed Ziyyat, Jean-Philippe Wolf, Francois Le Naour, Claude Boucheix, The molecular players of sperm–egg fusion in mammals, Seminars in Cell & Developmental Biology, 17, 2006, 254-263, www.elsevier.com/locate/sem/cdbd
- Shunichi Miyazaki and Masahiko Ito, Calcium Signals for Egg Activation in Mammals, J Pharmacol Sci, 100, 2006, 545-552, Published online in J-STAGE, DOI: 10.1254/jphs.CPJ06003X.
- Jan Tesarik and Ermanno Greco, The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology, Human Reproduction , 14, 1999, 1318-1323, http://www.ncbi.nlm.nih.gov/pubmed/10325285
Formative intermediate test
FORMATIVE INTERMEDIATE TEST 1- Date: 23.02.2024
- Type of test: quiz (multiple choice and true/false)
FORMATIVE INTERMEDIATE TEST 2
- Date: 01.03.2024
- Type of test: quiz (multiple choice and true/false)
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In order to achieve the specific training objectives of this course unit, students needs to have basic knowledge of Cytology, Cell Physiology and Cell Culture. Here you find the syllabus and teaching material (italian lessons) of the Remedial Course on Cell Physiology/Cell Culture.
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UNIT1
Introduction to the course and required prerequisites.
Laboratories on In Vitro Maturation (IVM) and In Vitro Fertilization (IVF) techniques.
Setting up a laboratory: basic equipment and facilities required for a laboratory of IVM and IVF.
Oocyte IVM in domestic animals:
- Tissue culture systems used for IVM. - Tissue culture media used for IVM. - Selection of ovaries retrieved from slaughterhouses. - Morphological evaluation of ovarian follicles and selection of cumulus-oocyte complexes (COCs). - Choosing and setting up of the best culture system for oocyte IVM in domestic animals. - Assessment of IVM oocytes.
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Risorsa
Here you find a brief description of the course "Biology of gametes, in vitro maturation (IVM) and in vitro fertilization (IVF) techniques"
The purpose of the course is to provide you with both the theoretical knowledge of the Biology and Physiology of mammalian gametes, and the practical skills needed to replicate in the laboratory two main steps of the reproductive process (the oocyte maturation and fertilization).
By using these techniques, it is possible to produce embryos in vitro.
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UNIT 2
IVF in domestic animals:
- Semen collection and assessment.
- Sperm preparation for IVF.
- Culture media for sperm capacitation and oocyte fertilization.
IVM and IVF procedures for in-vitro embryo production in the swine model
- Morphological evaluation and selection of in vitro matured oocytes.
- Semen preparation for IVF.
- Protocols for sperm capacitation and IVF.
- Morphological criteria and colorimetric assay for the evaluation of fertilized oocytes.
- IVF in Humans: introduction to FIVET and in vitro early embryo development.-
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Integrated file, contains new slides that have been used in class for active learning and discussions about IVM and IVF techniques, procedures and principles
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UNIT 3
IVM and IVF procedures for in vitro embryo production in the ovine model
Principles and simulation of IVF techniques used in the laboratory for assisted reproduction
- Principles about human IVM and superovulation protocols.
- Culture media for oocyte retrieval (ovum pick-up), sperm capacitation and IVF.
- Morphological evaluation of COCs collected by an ovum pick-up.
- Preparation of microdroplets under mineral oil.
- Protocols for sperm capacitation and IVF in the human model.
- Morphological evaluation of zygotes obtained by IVF.
- Introduction to assessment of early in vitro embryo development
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