Principles of IVF Laboratory Practice:

Optimizing Performance and Outcomes

(eBook PDF)
Visit to download the full and correct content document:
https://ebooksecure.com/download/principles-of-ivf-laboratory-practice-optimizing-perf
ormance-and-outcomes-ebook-pdf/
 Principles of IVF
Laboratory Practice

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:46:36, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 18:16:42, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 Contributors
Mina Alikani Nina Desai
Reproductive Science Center of New Jersey, Cleveland Clinic Fertility Center,
Eatontown, NJ, USA Cleveland, OH, USA
Basak Balaban Anick De Vos
Assisted Reproduction Unit, VKF Center for Reproductive Medicine,
American Hospital, Nisantasi-Istanbul, Brussels, Belgium
Turkey
Andrew D. Dorfmann
C. Brent Barrett Genetics & IVF Institute, Fairfax EggBank
Boston IVF, Boston, MA, USA
Caroline Drew
Dara S. Berger CARE Fertility, Nottingham, UK
UNC Fertility, Department of Obstetrics Thomas Ebner
and Gynecology, Division of Reproductive Kinderwunsch Zentrum Linz and Johannes
Endocrinology and Infertility, Raleigh, Kepler University Linz, Faculty of
NC, USA Medicine, Linz, Austria
Kathrin Berrisford Kara A. Ehlers
CARE Fertility, Nottingham, UK University of Louisville, KY, USA
Marcela Calonge Courtney Failor
IVI Santiago de Chile, Chile University of Texas Health Science Center,
San Antonio, TX, USA
Alison Campbell
CARE Fertility, Nottingham, UK Aisaku Fukuda
IVF Osaka Clinic, Japan
Tiencheng Arthur Chang
University of Texas Health Science Center, Giannoula Karagouga
San Antonio, TX, USA Dept. of Biomarker Discovery, Center of
Individualized Medicine, Mayo Clinic,
Greg L. Christensen Rochester, MN, USA
University of Louisville, KY, USA
Kathryn J. Go
Melicia Clarke-Williams IVF New England, a Boston IVF partner,
New York University Langone Medical Lexington, MA, USA
Center, New York, NY, USA
Cristina Hickman
Tyler Cozzubbo IVF Hammersmith, Ltd., Boston Place
Weill Cornell Medical College, Ronald Clinic, London, UK
O. Perelman and Claudia Cohen Center for
Heather S. Hoff
Reproductive Medicine, New York,
NY, USA UNC Fertility, Department of Obstetrics
and Gynecology, Division of Reproductive
Michael Crowe Endocrinology and Infertility, University of
Fertility Lab Sciences, Lone Tree, CO, USA North Carolina, Raleigh, NC, USA
ix
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:47:01, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 x Contributors

Rebecca Holmes Jonathan Lo

Boston IVF, Boston, MA, USA Yale Fertility Center, Yale University
School of Medicine, New Haven, CT, USA
Romain Imber
Centre Hospitalier Inter Régional Cavell Caroline McCaffrey
(CHIREC), Braine l’Alleud – Bruxelles, NYU Fertility Center, New York University
Belgium Langone Medical Center, New York,
NY, USA
Benedetta Iussig
GENERA Centres for Reproductive David H. McCulloh
Medicine, Rome, Marostica, Umbertide, NYU Fertility Center, New York University
Napoli, Italy Langone Medical Center, New York,
NY, USA
David Jareno
Centre Hospitalier Inter Régional Cavell Marius Meintjes
(CHIREC), Braine l’Alleud – Bruxelles, Frisco Institute for Reproductive Medicine,
Belgium Austin, TX, USA
Sangita K Jindal Satoshi Mizuno
Montefiore’s Institute for Reproductive IVF Osaka Clinic, Japan
Medicine and Health, Department
Obstetrics, Gynecology and Women’s Maximilian Murtinger
Health, Albert Einstein College of IVF Centers Prof Zech, Bregenz, Austria
Medicine, Bronx, NY, USA
Queenie V. Neri
Dawn A. Kelk Weill Cornell Medical College, Ronald
Yale University School of Medicine, Yale O. Perelman and Claudia Cohen Center for
Fertility Center, New Haven, CT, USA Reproductive Medicine, New York,
NY, USA
Sabine Kliesch
Centre of Reproductive Medicine and Verena Nordhoff
Andrology, University Hospital of Centre of Reproductive Medicine and
Münster, Germany Andrology, University Hospital of
Münster, Germany
María José de los Santos
IVF Laboratory Director of IVI, Oriol Oliana
Valencia, Italy IVF Hammersmith, Ltd., Boston Place
Clinic, London, UK
Patty Ann Labella
NYU Fertility Center, New York University Gianpiero D. Palermo
Langone Medical Center, New York, Weill Cornell Medical College,
NY, USA Reproductive Medicine,
Ithaca, NY, USA
Alex Lagunov
Fertility Lab Sciences, Lone Tree, CO, USA Thomas B. Pool
Fertility San Antonio, TX, USA
Georgios Liperis
Westmead Fertility Centre and Institute of Nicolas Prados
Reproduction, University of Sydney, IVF Laboratory Director of IVI,
Westmead, Australia Valencia, Italy

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:47:01, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 Contributors xi

Pooja Rambhia Jun Tao

Case Western Reserve University School of Fertility Treatment Center, Tempe,
Medicine, Cleveland, OH, USA AZ, USA
Michael L. Reed Stephen Troup
Center for Reproductive Medicine of New Hewitt Fertility Centres, Liverpool
Mexico, Albuquerque, NM, USA Women’s Hospital, Liverpool, UK
Laura Rienzi Filippo M. Ubaldi
GENERA Centers for Reproductive GENERA Centres for Reproductive
Medicine, Napoli, Italy Medicine, Rome, Marostica, Umbertide,
Napoli, Italy
Mitchel C. Schiewe
Ovagen Fertility, Newport Beach, Pierre Vanderzwalmen
CA, USA Centre Hospitalier Inter Régional Cavell
(CHIREC), Braine l’Alleud – Bruxelles,
Karen Schnauffer Belgium
Hewitt Fertility Centres, Liverpool
Women’s Hospital, Liverpool, UK Sabine Vanderzwalmen
IVF Centers Prof Zech, Bregenz, Austria
Cecilia Sjöblom Centre Hospitalier Inter Régional Cavell
Westmead Fertility Centre and Institute of (CHIREC), Braine l’Alleud – Bruxelles,
Reproduction, University of Sydney, Belgium
Westmead, Australia
Shunping Wang
Amy E.T. Sparks Brown University, Providence, RI, USA
University of Iowa Carver College of
Medicine, Department of Obstetrics and Klaus E. Wiemer
Gynecology, Iowa City, IA, USA Poma Fertility, Kirkland, AZ, USA
Jason E. Swain Barbara Wirleitner
Fertility Lab Sciences, IVF Centers Prof Zech, Bregenz, Austria
Lone Tree, CO, USA

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:47:01, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:47:01, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 Foreword
Soon we shall reach the 40th anniversary of the birth of Louise Brown, the world’s first ‘test-
tube’ baby, born in a small town in the north of England, thanks to the tireless work of
Robert Edwards and Patrick Steptoe. During the intervening years, it has been wonderful to
see how the technologies associated with the treatment of human infertility have developed
and how widely they have been adopted around the world. It is estimated that around six
million in vitro fertilization (IVF) children have now been born worldwide, a number that
continues to increase steadily.
The IVF laboratory has changed dramatically over the intervening four decades and is
consequently far more sophisticated with regards to the technologies used routinely to treat
infertile couples, which now include intra-cytoplasmic sperm injection (ICSI), embryo
biopsy, pre-implantation genetic screening, blastocyst culture, vitrification and time-lapse
analysis. In parallel, there has been a rapid increase in the number of clinics worldwide.
Consequently, this has created a pressing need for the standardization of training and
education for embryologists.
In this book, Montag and Morbeck have assembled contributions from several experts
around the world, who have penned detailed procedures for each component of IVF – from
the very inception of a laboratory through to the very conception of a pregnancy. This book
therefore represents a veritable gold mine of procedures which will be invaluable not only to
new laboratories but also to all those clinics that strive to make our patients’ dreams of
having a family fulfilled, and hence a copy should be located in every clinic worldwide.

Professor David K. Gardner

School of BioSciences
The University of Melbourne
Parkville, Victoria
Australia

xiii
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 18:17:22, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 18:17:22, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 Preface
Since the first successful human in vitro fertilization (IVF) treatment in 1978, IVF has
transformed from an experimental procedure to an established standard of care that is
practiced throughout the world, every year helping thousands of infertile patients realize
their dream of parenthood. We can deliver this incredible care only by standing on the
shoulders of the pioneers of our field, who through trial, error and determination developed
the foundation for how we practice today. We are fortunate to work in a field that rapidly
advances, and the IVF laboratory, at the core of this advancement, has become increasingly
complex. This complexity is not limited to the procedures themselves but starts during the
planning and construction of an IVF laboratory and continues with the training of young
embryologists. Operation of an IVF laboratory comprises a portfolio of techniques that
cover basic as well as advanced practices. Capturing and transforming this breadth of
material into a practical, cookbook-like manual seemed to be obvious, and we, the editors,
were encouraged that perhaps this is a gap that needs filling.
Today’s embryology world is rapidly changing, and with it come new challenges as new
generations of embryologists begin their careers. As the pioneers of IVF transition to
retirement, all the knowledge they acquired through learning by doing has yielded what
we consider to be the Principles in IVF Practice – sound procedures that will work in almost
every laboratory. It is our aim to capture these principles for all those who enter our
expanding and fast-growing field and do not have the chance or the freedom to learn by
trial and error. Many embryologists in laboratories around the globe use the basics that are
described in this book. One can consider these methods as a foundation of good working
practice for new embryologists.
One theme of this book is the mix of contributing authors that come from different
parts of the world with very different points of view. Thus, experienced practitioners in the
world of IVF can compare their own and established concepts with those of others and
perhaps learn something new as well. We sincerely hope that this book will contribute to
and stimulate discussions in many laboratories on how to perform or adapt procedures.
We, too, learned much during the editing process and are eager to place the printed version
on our laboratory bench.

xv
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:52:10, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:52:10, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238
 Section 1 Starting a New Laboratory and Training Protocols

Establishing and Equipping a

Chapter

1 New IVF Laboratory

Alex Lagunov, Michael Crowe and Jason E. Swain

Introduction
Attention to detail and robust quality control systems are essential components for a
successful in vitro fertilization (IVF) laboratory, necessary to maintain high success rates.
This not only pertains to daily laboratory operations, such as adequate staff training,
monitoring of equipment function and weekly/monthly success rates, but also entails
proper laboratory design and construction. A highly successful IVF laboratory requires
careful consideration of layout and workflow and use of appropriate materials to avoid
introducing potentially embryotoxic factors into the laboratory environment.

1.1 Background
The design and equipping of a new IVF laboratory can be a daunting and tedious endeav-
our. Numerous factors must be considered, including space requirements to accommodate
staff and equipment as well as layout for optimal workflow. Proximity to other rooms, such
as medical gas storage, operating/procedure rooms and transfer rooms, as well as IVF
storage is also relevant. The potential for future laboratory expansion is also often desirable.
Thus, initial planning of space requirements should consider these items. Each facility will
have its own unique demands that will affect the ultimate size and layout of the laboratory
space. However, commonalities exist between highly successful laboratories in terms of how
the laboratory is built and equipped. Furthermore, key steps are often taken prior to clinical
use to ensure proper functioning of the built space.
The following are abbreviated procedures addressing key components to ensure proper
establishment and equipment of a modern IVF laboratory. Many key components focus on
use of appropriate materials and systems to ensure proper air quality and growth condi-
tions. Of paramount importance is constant oversight during the construction process to
ensure that key details are met. Once these requirements have been met, diligence and a
thorough quality management programme are required to ensure that optimal laboratory
conditions remain intact.

1.2 Supplies and Equipment

 A professional team of architects and a general contracting firm experienced in medical
facility build-out
 A penthouse suite or stand-alone building, allowing for proper laboratory layout
 A functional laboratory/clinic design plan that is approved by the laboratory director
and architectural and construction teams

1
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 2 Alex Lagunov, Michael Crowe and Jason E. Swain

: Careful attention to required workflow, equipment, electrical and information

technology (IT) needs

 Appropriately chosen construction materials

: Low or no volatile organic compounds (VOCs)
– Flooring /adhesives – sheet vinyl with welded seems, low-VOC adhesive
– Insulation – avoid around perimeter of laboratory; formaldehyde free product or
mineral wool if required
– Paint – no-VOC latex paint product for all laboratory spaces
– Cabinetry – powder-coated/painted-metal cabinetry, no particle board or laminate
– Countertops – Corian, Trespa or some other low-VOC hard material without
adhesives
– Furniture – medical- or laboratory-grade furniture with vinyl covering and low-
VOC materials
– Avoid use of printers, corkboards, xylene markers inside the laboratory space
– Copper piping for gas lines with brazed connection; stainless steel piping also
appropriate
– Tyvek wrap around lab perimeter behind drywall to help maintain positive pressure

: Minimum 36-inch doors for equipment installation - with gaskets and sweeps
: Security system on entry doors (lockable)
: Gas manifold system – gas regulators, Tygon tubing
– Automatic tank switchers may be used but are not required.
– Regulators to measure tank pressures are needed at the gas source.
– Low-pressure regulators at the gas outlet are recommended to meet varying
pressure requirements of different incubator models.

: Required laboratory equipment/vendor list

– Microscopes, micromanipulators, centrifuges, laminar flow hoods, incubators,
refrigerators, alarm system, computers, consumables, etc.

: Backup generator allowing for all equipment to be supplied with uninterrupted power
– Natural gas or diesel – may be dictated by code or building requirements. A minimum
of eight-hour backup is recommended, and fuel tank size should be appropriate.
Battery backups may be used for some equipment, but must be adequate to supply
several hours of power.

: Appropriate, dedicated heating, ventilation and air-conditioning (HVAC) system

– VOC filtration (various systems exist that use combinations of UV light, carbon
filters and potassium permanganate)
– High-efficiency particulate air (HEPA) filtration
– properly sized and designed unit to maintain positive pressure inside the laboratory
– proper placement of air vents to avoid being directly over critical equipment

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 Establishing and Equipping a New IVF Laboratory 3

: Cleaning products
– 3% Hydrogen peroxide solution or commercially available low-VOC, embryo-
safe cleaning solutions
: Required laboratory manuals
– Quality management programme, protocols and policies, equipment
maintenance, safety (Material Safety Data Sheets), etc.

1.3 Quality Control

 Keep a record of meeting minutes and plan revisions with construction company and
contractors to track discussions.
 Continue to monitor the construction process on a daily/weekly basis to ensure that
design and specifications are followed.
: Ensure the use of low-VOC products in full range of materials used to construct the
laboratory (and procedure room if in proximity to the laboratory).
 Air quality testing prior to and after construction is recommended to achieve appropriate
laboratory air quality (measurement of VOCs in ppb levels is recommended).
 Perform a mouse embryo assay (MEA) or approved bioassay prior to clinical cases.

1.4 Procedure
1.4.1 Initial Project Planning
1. Identify a location that suits the needs of the clinic/laboratory.
a. Proximity to an existing clinic is recommended.
b. Must be adjacent to procedure room/operating room (OR) and embryo transfer room.
c. Adequate laboratory space for offices, storage, equipment, specimen collection,
medical gasses, etc.
2. Locate and interview several construction and architectural companies based on
previous experience in medical facility design and build-out in order to ensure that
quality standards are clearly understood and met.
3. Prepare a laboratory floor plan considering workflow, including all laboratory
equipment, maintaining close proximity between intracytoplasmic sperm injection
(ICSI) stations and other IVF workstations to all incubators.
a. Compile equipment list and plan locations.
b. Plan placement of lights, air vents and electrical and IT outlets appropriately.
c. Design a separate dedicated medical gas/liquid nitrogen storage room. It is
recommended to be a sound proof, well-ventilated room located in close proximity
to a freight elevator or delivery area, if possible. If feasible, locating this room near
IVF liquid nitrogen or vapour tank storage area with vacuum jacketed lines for
filling is recommended.
i. Ensure that building regulations allow for use and transport of high-pressure
medical gasses and liquid nitrogen.
d. Design a separate room for consumables storage and off-gassing.
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 4 Alex Lagunov, Michael Crowe and Jason E. Swain

4. Once plans are approved and construction is initiated, ensure that HVAC is designed
based on the required square footage and filtering capacity and meets requested
standards (discussed further in HVAC).
5. Ensure that a natural gas or diesel backup power generator is included in the laboratory
design and is sufficient to support more than eight hours of backup power supply. Set
up generator service schedule to include initial two-hour load bank test, as well as semi-
annual maintenance/inspections and yearly load tests.
a. If a generator is not feasible, uninterruptible power supply (UPS) units are required
on all crucial equipment with supply long enough to permit corrective action in case
of power failure.
6. Fill out and submit required laboratory applications and paperwork to the relevant
governing agencies.

1.4.2 Build-Out Process

1. Verify that all materials used are low or no VOCs (verify during onsite inspections).
a. Insulation: Insulation is often used in construction. However, chemicals such as
formaldehyde are often present. Presence of insulation on the direct periphery of the
IVF laboratory should be avoided if possible. If unable to avoid due to the presence
of an outside wall, use of a formaldehyde-free product is recommended. Alternatives
for sound dampening include mineral wool.
b. Flooring: Welded instead of glued seems of sheet vinyl flooring are recommended
for sanitary reasons as well as minimal VOC release. Prior to clinical use, floors need
to be cleaned with appropriate cleaners, such as hydrogen peroxide, along with
simple regular sweeping, vacuuming or steam mopping. Avoid the use of any
potentially harmful cleaners or waxes inside the laboratory.
c. Paints: Latex-based no-VOC paints should be used on all painted surfaces.
d. Cabinetry: Powder-coated metal cabinetry or stainless steel is suggested to avoid
concerns with laminate and particle board, which would likely contain adhesives with
high levels of VOCs.
e. Furniture: Purchase all medical- or laboratory-grade chairs with vinyl-coated
seating surfaces that are easily cleaned of any contamination. Tables and countertop
should be made of either Trespa, Corian, stainless steel or some other hard material
devoid of adhesives (laminate).
f. Cleaners: Alcohol is discouraged due to VOCs. 3% Hydrogen peroxide–based
cleaning solutions are recommended.
2. Ensure that HVAC ductwork uses cleaned steel instead of plastic or other materials.
a. Ductwork should be cleaned onsite by the installation crew using alcohol. Swabs can
be taken to verify cleanliness and absence of oil and dust, etc.
b. Verify that ductwork is sealed during the construction process to avoid dust and
contaminants..
3. Ensure that sufficient gas supply is provided for all planned equipment inside the IVF
laborator.
a. Verify that gas piping is in the correct location.
b. Verify that proper manifold system and regulators are present and functional.
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 Establishing and Equipping a New IVF Laboratory 5

4. Ensure proper placement and functioning of various items.

a. Confirm placement of light switches and outlets and all necessary equipment for
performing daily operations.
b. Confirm the placement and swing of doors, proper locks, seals, sweeps, etc.
5. Verify function of the generator. If a power outage creates a lag prior to generator power
surge, ensure that all equipment is plugged into the UPS.
6. If windows are present, ensure that blackout blinds are available to help regulate light/
heat within the laboratory. Incubators and ICSI workstations should be protected from
direct sunlight to avoid potential issues with light and/or temperature regulation.
7. Confirm the absence of cork boards, printers/toners or unapproved markers containing
xylene within the confines of the laboratory during move-in. These items may be kept in
a separate laboratory office area away from embryo culture and handling locations.

1.4.3 HVAC
1. Select an HVAC company.
a. Decide on a commercially available ‘plug-and-play’ filtration system or have HVAC
company built from scratch based on required IVF laboratory air quality demands.
System should be dedicated to the IVF laboratory.

i. Filtration should include pre-filters (MERV) and HEPA filtration to remove

particulates. Importantly, filtration should also reduce VOC content and can
include use of ultraviolet (UV) light for photo-catalytic oxidation or use of
chemical filter beds with carbon and potassium permanganate mixtures.
ii. Placement of filtration system is important; it should be as close to the outlet air
ducts in the laboratory as possible to avoid risk for introduction of contaminants
in-line after the filters.
iii. Ensure that air supply duct placement is not directly above/near ICSI stations
or incubators, as this will affect temperature regulation of the stage
warming units.

b. Ensure that only metal ductwork is used. This should be pre-cleaned and sealed
during construction to avoid introduction of contamination.
c. Achieve positive pressure in the laboratory.
i. Request Tyvek wrapping of the laboratory, hard deck ceiling, gasketted lights and
doors and door sweeps.
d. Use proper air exchange rates and ratio of outside-to-inside air to achieve desired air
quality.
i. Recommendations vary and will depend on the HVAC system and beginning air
quality.
e. Ensure a proper water supply drawn to the HVAC unit to allow for constant
humidity control.
f. Ensure that a pressure display panel is installed inside the laboratory to monitor and
record positive pressure for proper system functionality as per daily laboratory
quality control.
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 6 Alex Lagunov, Michael Crowe and Jason E. Swain

g. Set up a regular maintenance contract with an appropriate company for servicing

the belts and other items of the HVAC unit as well as all necessary filter changes to
allow for initially targeted air quality standards.

1.4.4 Equipment
1. Determine volume estimates for the laboratory, and obtain quotes for selected
equipment.
a. Quotes and equipment specs (size, electrical requirements, heat output) should be
obtained early on in the design process.
i. Backup units of critical equipment should be considered (ICSI microscopes,
incubators, fridges, freezers, etc.).
b. Low-oxygen incubators are mandatory.
i. Mix of bench-top units and box-type incubators is recommended.
ii. Low oxygen may be supplied via premixed tanks or through use of separate
nitrogen and carbon dioxide sources depending on type of incubator.
(1) Nitrogen may be supplied through gas cylinders, high-pressure liquid
nitrogen tanks or via a nitrogen generator. Cost, ease of delivery and usage
are all factors to consider.
iii. Use in-line VOC gas filters between gas supply and incubators – change out
every four to six months.
2. Attempt to purchase equipment from vendors that are able to service the items
within a short amount of time if equipment failures arise.
3. Set up a regular laboratory equipment maintenance system with local service
providers (hoods, centrifuges, pipette calibration/validation).
4. Ensure that all incubators and cryo-storage vessels are monitored by an alarm with
the ability to e-mail and/or call in case of an emergency.
a. Wireless systems are now available to help with equipment movement and
location and are recommended.

1.4.5 Burn-In
1. After construction, the laboratory should be wiped down with an approved disinfectant/
cleaner.
a. Walls, floors, cabinets and other surfaces should be wiped several times over the
course of the burn-in period.
2. Equipment should be moved into the laboratory and cleaned/tested prior to clinical use.
a. Of critical importance is burn-in of laboratory incubators. Incubators should be cleaned
according to manufacturer’s instructions and temperature raised to greater than 45°C
for several days or up to two weeks to promote off-gassing, cleaned and tested.1
b. Laboratory temperature can be raised to help with off-gassing of other
laboratory items.
c. Verify correct temperature, gas and functioning of all equipment for several days
after burn-in on quality control sheets to verify proper environmental conditions
and functioning.
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 Establishing and Equipping a New IVF Laboratory 7

3. Perform pH testing of culture media used for embryos and gametes with the aim of
achieving desired pH based on incubator carbon dioxide concentration.1–3
4. Perform air quality testing measuring VOCs and particle counts before and after burn-
in, and record the differences in the readings for quality control records.
a. This can be performed by a professional company or by laboratory personnel if
hand-held particle counters and VOC meters have been purchased by the laboratory
or can be obtained from vendors. Sensitivity of these units should be considered
(parts per billion recommended for VOC assessment).
5. Perform a MEA to ensure proper incubator conditions.
a. All culture supplies should also be pre-tested with an appropriate bioassay prior to
clinical use.

1.5 Notes
A. Rationale behind using low-VOC materials and a dedicate HVAC system is based on
various publications suggesting the cytotoxicity of VOCs.4–7 VOCs include various
types of alkanes, aldehydes, ketones and alcohols commonly concentrated at high levels
in paints, glues and flooring.
B. After laboratory commissioning, ongoing maintenance and quality control are required
to ensure optimal function and culture conditions.
i. Schedule for HVAC filter changes, routine cleaning and disinfecting of the
laboratory, daily monitoring of incubator gas and temperature, testing of generator
and alarm systems, etc.
ii. Monitoring of key performance indicators (KPIs) to identify possible issues
(1) Fertilization rates, embryo development rates, clinical pregnancy, etc.

References 4. Hall, J., et al. The origin, effects and control

1. Swain, J. and Lagunov, A. IVF incubator
handling, in Standard Operational
Procedures in Reproductive Medicine
Laboratory and Clinical Practice, ed.
B. Rizk and M. Montag. (Boca Raton,
FL: CRC Press, in press).
2. Swain, J. E. Optimizing the culture
environment in the IVF laboratory: impact
of pH and buffer capacity on gamete and
embryo quality. Reprod Biomed Online
2010; 21(1):6–16.
3. Swain, J. E. Is there an optimal pH for
culture media used in clinical IVF? Hum
Reprod Update 2012; 18(3):333–9.
of air pollution in laboratories used for
human embryo culture. Hum Reprod 1998;
13(Suppl. 4):146–55.
5. Schimmel, T., et al. Removal of volatile
organic compounds from incbuators used
for gamete and embryo culture. Fertil Steril
1997; 68(Suppl. 1):S165.
6. Morbeck, D. E. Air quality in the assisted
reproduction laboratory: a mini-review.
J Assist Reprod Genet 2015; 32(7):1019–24.
7. Heitmann, R. J., et al. Live births achieved
via IVF are increased by improvements in
air quality and laboratory environment.
Reprod Biomed Online, 2015.

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:35, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.003
 Section 1 Starting a New Laboratory and Training Protocols

Basic Embryology Skills in the

Chapter

2 IVF Laboratory
Stephen Troup and Karen Schnauffer

Introduction
Before aspiring embryologists can even begin to perform the procedures that will comprise
the majority of their routine workload, there are a number of essential basic skills that
underpin their day-to-day work. Failure to acquire these basic skills at the outset can lead to
an inability to perform even the simplest of tasks appropriately, an inability to troubleshoot
problems and even the potential to place gametes, embryos and patients at risk. As such,
considerable time and effort should be dedicated to the acquisition of these basic skills, and
this is often reflected in the formally recognised embryology training schemes which now
exist (e.g. European Society of Human Reproduction and Embryology (ESHRE) Embry-
ology Certification). A competent embryologist will also equip himself or herself with an
appropriate level of theoretical knowledge to allow an understanding of the rationale for
processes and procedures. The fundamental nature of the work that an embryologist
performs requires that all aspects of training be completed to the highest possible standard,
and attention to detail must persist throughout. The importance and relevance of the basic
skills described in this chapter should not be underestimated.

2.1 Confidentiality
It is a fundamental rule in medical ethics that information given by, or relating to, a
patient remains private and that it should only be shared with a third party under certain
agreed circumstances and more often than not with the permission of the patients
themselves. In many countries, regulation determines the extent of patient confidentiality.1
In the absence of such guidance, it remains the responsibility of the embryologist to give
due consideration to the often extremely private and personal information that is made
available to him or her during a patient’s treatment in order to perform his or her tasks
and, in particular, with whom this information should be shared. Assumptions should not
be made that any third party to whom information is given will respect the confidential
nature of the information, and involving the patient in any such disclosure is always
advisable.

2.2 Record Keeping

It goes without saying that accurate record keeping within an embryology laboratory is of
paramount importance – but it is all too often overlooked. The following set of basic rules,
which seek to minimise risk associated with record keeping, should be adopted by embry-
ologists in all areas of their work.
8
Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:00, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.004
 Basic Embryology Skills in the IVF laboratory 9

1. Any records pertaining to a patient should be identified using three identifiers, such as
forename(s) and surname(s), the patient’s date of birth and address and, importantly, a
unique identifier such as the hospital/clinic number.
2. All written records should be made in black ink.
3. All entries in the patient record should be signed by the embryologist making the record,
together with the date and time of the entry and the designation of the embryologist.
4. All written records should be clearly legible, and where a record needs to be
corrected, this should be done by a single line through the incorrect entry (so as not
to completely obscure the original entry), which then should be signed and dated as
described in rule 3. Correction fluid should not be used.
5. Electronic patient records (EPRs) should be backed up regularly and securely.
6. Paper documents should be scanned into an EPR in such a way that the original
document can be reconstituted.

2.3 Traceability
Traceability of gametes, embryos and everything else that comes into contact with them (i.e.
consumables, culture media and equipment) for the duration of the treatment is essential
and relies on robust recording keeping, as described earlier, to ensure that the correct
identity of patients, gametes and embryos is maintained at all times. This enables gametes
and embryos to be accurately tracked throughout treatment from procurement to replace-
ment, storage or being discarded. In the event of a commercial product being withdrawn,
the traceability system would identify which patients had been affected by the faulty product
and enable a full investigation to be carried out. Below is a list of traceability points that
should be included in a traceability system:
1. Centres should ensure that patients and partner(s)/donor(s) can be uniquely and
accurately identified.
2. The treatment type and date should be clearly documented.
3. Any container used for culture of embryos or preparation of gametes should be fully
labelled with the patient’s details.
4. All equipment and materials coming into contact with a patient’s gametes or embryos
should be identified with a product description, source and batch numbers of the
product used.
5. The centre from which the gametes or embryos have been created should be recorded.

2.3.1 Aseptic Technique

Successful culture of gametes and embryos in vitro depends on keeping the cultures free from
contamination by micro-organisms such as fungi, bacteria and viruses. Within an assisted-
conception laboratory, many potential sources of biological contamination exist, including the
air within the laboratory, non-sterile supplies, unclean surfaces, equipment and clothing and also
the individuals who work within the laboratory! In some countries, regulations exist specifying
the ‘quality’ of air required within laboratories where gametes and embryos are handled.2
‘Aseptic technique’ is a set of procedures designed to create a ‘barrier’ between micro-
organisms in the environment to reduce the probability of contamination from these
sources. There are four basic elements of aseptic technique, namely, a sterile work area,
good personal hygiene, sterile reagents and sterile handling techniques.

Downloaded from https:/www.cambridge.org/core. Boston University Theology Library, on 29 May 2017 at 17:14:00, subject to the Cambridge
Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/9781316569238.004
Another random document with
no related content on Scribd:
 7.

Who tooke to wife as yee shall vnderstand

A mayden of a noble house and olde,
Raulfe Neuil’s daughter, earle of Westmerland,
Whose sonne earle Richard, was a baron bolde,
And had the right of Salisbury in holde,
Through mariage made with good earle Thomas heyre,
Whose earned prayses neuer shall appayre.

8.

The duke my father had by this his wife

Four sonnes, of whom the eldest Edward hight,
The second Eadmund, who [in youth] did loose his life,
[1174]
At Wakefielde slayne by Clyfford cruell knight:
I George am third, of Clarence duke by right:
The fourth, borne to the mischiefe of vs all,
Was duke of Glocester,[1175] whom men did[1176]
Richard call.

9.

Whan as our sire in sute of right was slaine,

(Whose life and death himselfe declared earst)
My brother Edward plyed his cause amayne,[1177]
And got[1178] the crowne, as Warwicke hath rehearst:
The pride whereof so deepe his stomacke pearst
That hee forgot his friendes, dispisde his kin,
Of oth or office passing not a pyn.[1179]

10.

Which made the earle of Warwicke to maligne

My brother’s state,[1180] and to attempt a way
To bring from prison Henry, seely king,
To helpe him to the kingdome[1181] if hee may,
And knowing mee to bee the chiefest stay
My brother had, hee did mee vndermine
To cause mee to his treasons to encline.[1182]

11.

Whereto I was prepared long before,

My brother had beene to mee so vnkinde:
For sure no cankar fretteth flesh so sore,[1183]
As vnkinde dealing doth a louing minde:
Loue’s strongest bandes vnkindnes doth vnbinde,
It moueth loue to malice, zeale to hate,
Chiefe friendes to foes, and brethren to debate.

12.

And though the earle of Warwicke, subtile sire

Perceiude I bare a grudge against my brother,
Yet toward his feate to set mee more on fire,
Hee kindled vp one firebrand with another:
For knowing fancy was the forcing rother
Which stirreth youth to any kinde of strife,
Hee offered mee his daughter to my wife.

13.

Where through, and with his crafty filed tongue,

Hee stale my heart that earst vnsteady was,
For I was witlesse, wanton, fond and yong,
Whole bent to pleasure, brittle as the glasse,
I can not lye, In vino veritas:
I did esteeme the bewty of my bryde
Aboue my selfe, and all the world beside.

14.

These fond affections ioynd with lacke of skill,

(Which trap the heart, and blind the eyes of youth,
And pricke the minde to practise any ill)
So tickled mee, that voyde of kindly truth,
(Which if it want all wretchednes[1184] ensueth)
I stinted not to persecute my brother,
Til time hee left his kingdom to another.

15.

Thus carnall loue did quench the loue of kinde,

Till lust were lost through fancy fully fed:
But whan at length I came vnto my minde,
I saw how lewdly lightnes had mee led,
To seeke with payne the perill of my heade:
For had king Henry once beene setled sure,
I was assurde my dayes could not endure.

16.

And therefore, though I bound my selfe with[1185] oth

To help king Henry all that euer[1186] I might,
Yet at the treaty of my brethren both,
Which reason graunted to require but right:
I left his part, whereby hee perisht quite:
And reconcilde mee to my brethren twayne,
And so came Edward to the crowne agayne.

17.

This made my father[1187] in law to fret and fume,

To stamp and stare, and call mee false forsworne,
And at the length with all his power, presume
To help king Henry, vtterly forlorne:
Our friendly proffers still hee tooke in scorne,
Refused peace, and came to Barnet fielde,
And there was kild, because hee would not yeelde.
 18.

His brother also there with him was slayne,

Whereby decayed the keyes of chiualrye:
For neuer liu’d the matches of them twayne,
In manhood, power, and martiall pollecy,
In vertuous thewes, and friendly constancy,
That would to God, if it had bene his will,
They might haue tournde to vs and liued still.

19.

But what shalbe, shall bee: there is no choyse,

Thinges needes must driue as desteny decreeth,
For which wee[1188] ought in all our haps reioyce,
Because the eye eterne all things foreseeth
Which to no ill at any tyme agreeth,
For ills, to ill to vs, be good to it,
So far his skill’s exceede our reach of wit.

20.

The wounded man which must abyde the smart

Of stitching vp, or searing of his sore,
As thing to bad, reproues the surgeon’s art
Which not withstanding doth his health restore.
The childe likewise to science plied sore,
Counts knowledge ill, his teacher to be wood,
Yet surgery and sciences be good.

21.

But as the pacient’s griefe and scholer’s payne,

Cause them deme bad such things as sure be best,
So want of wisdome causeth vs complaine
Of euery hap, wherby we seme opprest:
The poore doe pine for pelfe, the rich for rest,
And when as losse or sicknesse vs assayle
 We curse our fate, our fortune we bewayle.

22.

Yet for our good, God worketh euery thing:

For, through the death of these two noble peres,
My brother liu’d and raynde a quiet king,
Who, had they liued, perchaunce in course of years
Would haue deliuered Henry fro the breres,
Or holpe his sonne t’[1189]enioy the carefull crowne,
Wherby our line should haue bene quite put downe.

23.

A carefull crowne it may be iustly named,

Not onely for the cares thereto annext,[1190]
To see the subiect well and duly framed,
With which good care few kings are greatly vext,
But for the dred wherwith they are perplext,
Of losing lordship, liberty, or life:
Which wofull wracks in kingdoms happen ryfe.

24.

The which to shun while some to sore haue sought,

They haue not sparde all persons to suspect:
And to destroy such as they gilty thought,
Though no apparaunce proued them infect.
Take me for one of this wrong punisht sect,
Imprisonde first, accused without cause,
And done to death, no processe had by lawes.

25.

Wherin I note how vengeaunce doth acquite

Like yll for yll, how vices vertue quell:
For as my mariage loue did me excite
Agaynst the king my brother to rebell,
So loue to haue his children prosper well,
Prouoked him, agaynst both law and right,
To murder me, his brother, and his knight.

26.

For by his queene two princelyke sonnes he had,

Borne to be punisht for their parent’s synne:
Whose fortunes kalked made the father sad,
Such wofull haps were found to be therin:
Which to auouch, writ in a rotten skin,
A prophesie was found, which sayd, a G
Of Edward’s children should destruction bee.

27.

Mee to bee G, because my name was George,

My brother thought, and therefore did mee hate,
But woe be to the[1191] wicked heads that forge
Such doubtfull dreames to breede vnkinde debate:
For God, a gleue, a gibbet, grate, or gate,
A Gray, a Griffeth, or a Gregory,
As well as George, are written with a G.

28.

Such doubtfull riddles are no prophesies:

For prophesies, in writing though obscure,
Are playne in sence, the darke be very lies:
What God foresheweth is euident and pure,
Truth is no harold nor noe sophist sure:
She noteth not men’s names, their shieldes, nor
creasts,
Though she compare them vnto byrds and beasts.

29.

But whom she doth forshewe shall rayne by force,

She tearms a wolfe, a dragon, or a beare:
A wilfull prince, a raynlesse ranging[1192] horse:
A bold, a lion: a cowarde much in feare,
A hare or harte: a crafty, pricked eare:
A leacherous, a bull, a goate, a foale:
An vndermyner, a moldwarpe, or a mole.

30.

By knowen beastes thus truth doth playne declare

What men they be of whom shee speakes before:
And who so can men’s properties compare
And marke what beast they doe resemble more:
Shall soone discerne who is the griesly bore:
For God by beastes expresseth men’s condicions,
And not theyr badges, haroldes supersticions.

31.

And learned Merlyne, whom God gaue[1193] the sprite

To know and vtter princes actes to come,
Like to the Iewish prophets, did recite
In shade of beastes, theyr doings all and some,
Expressing plaine by maners of the dome,
That kinges and lordes such propertyes should haue
As haue the beastes whose name he to them gaue.

32.

Which while the foolish did not well consider,

And seeing princes gaue, for difference
And knowledge of theyr issues mixt together,
All maner beastes for badges of pretence,
There tooke those badges to expresse the sence
Of Merlyne’s minde, and those that gaue the same,
To bee the princes noted by theyr name.

33.
And hereof sprang the false namde prophesies,
That goe by letters, siphers, armes, or sines:
Which all bee foolish, false, and crafty lyes,
Deuisde by gesse, or guiles vntrue deuines:
For whan they saw that many[1194] of many lynes
Gaue[1195] armes alyke, they wist not which was hee
Whom Merlyne ment the noted beast to bee.

34.

For all the broode of Warwicke’s gaue the beare,

The Buckinghams doe likewise gieue the swan:
But which beare bearer should the lyon teare
They were as wise as Goose the fery man:
Yet in theyr skill they ceased not to scan,
And to bee deemed of the people wise,
Set forth theyr gloses vpon[1196] prophesies.

35.

And whome they douted openly to name

They darkely tearmed or by some letter ment,
For so they thought, how euer the world did frame,
Preserue themselues from shame, or being shent:
For, howsoeuer contrary it went,
They might expound their meaning otherwise,
As haps in things should newely still arise.

36.

And thus there grewe of a mistaken truth,

An art so false as made the true suspect:
Whereof hath come much mischiefe, more the ruth
That errours should our mindes so much infect,
True prophets[1197] haue fowly beene reiect:
The false, which breede both murder, warre, and strife,
Beleeued to the losse[1198] of many a good man’s life.
 37.

And therefore, Baldwine, teach men to discerne,

Which prophecies be false and which bee true:
And for a ground this lesson let them learne,
That all bee false which are deuised newe:
The age of thinges are iudged by the hue:
All riddels made by letters, names or armes,
Are yong and false, far worse then witche’s charmes.

38.

I knowe thou musest at this lore of mine,

How I, no studient, should haue learned it:
And dost impute it to the fume of wine
That stirs the tongue, and sharpneth vp the wit:
But harke, a friend did teach mee euery whit,
A man of mine, in all good knowledge rife,
For which hee guiltlesse lost his learned life.

39.

This man abode my seruaunt many a day,

And still in study set his whole delight:
Which taught mee more then I could beare away
Of euery arte: and by his searching sight
Of thinges to come hee would foreshew as right,
As I rehearse the pageants that were past:
Such perfectnes God gaue him at the last.

40.

He knew my brother Richard was the bore,

Whose tuskes should teare my brother’s boyes and me,
And gaue me warning therof long before:
But wyt nor warning can in no degree
Let thinges to hap, which are ordainde to bee:
Witnesse the painted lionesse, which slue
 A prince imprisoned, lyons to eschewe.

41.

He told me eke[1199] my yoke fellow should dy,

(Wherin would God he had bene no deuyne)
And after her death I[1200] should woo earnestly
A spouse, wherat my brother would repine,
And finde the meanes she should be none of[1201] mine:
For which such malice should among vs ryse,
As saue my death no treaty should decise.

42.

And as he sayd, so all things came to passe:

For whan king Henry and his sonne were slaine,
And euery broyle so throughly quenched was
That the king my[1202] brother quietly did raygne,
I, reconciled to his loue agayne,
In prosperous health did leade a quiet lyfe,
For fiue yeares space with honours laden rife.

43.

And to augment the fulnesse of my blisse,

Two louely children by my wife I had:
But froward hap, whose maner euer is
In chiefest ioy to make the happy sad,
Bemixt my sweete with bitternes too bad:
For while I swam in ioyes on euery side,
My louing wife, my cheifest iewell dyed.

44.

Whose lacke whan sole I had bewaylde a yeare,

The duke of Burgoine’s wife, dame Margarete,
My louing sister willing me to chere,
To mary[1203] agayne did kindely me intreate:
And wisht me matched with a mayden nete,
A step daughter of her’s, duke Charles’ hayre,[1204]
A noble damsell, yong, discrete and fayre.

45.

To whose desire because I did enclyne,

The king my brother douting my degree
Through prophesies, against vs did repyne,
And at no hand would to our wills agree:
For which such rancoure pearst both him and mee,
That face to face we fell at flat defiaunce,
But were appeased by frends of our aliaunce.

46.

Howbeit my mariage vtterly was dasht:

Wherin because my seruant sayd his minde,
A meane was sought wherby he mought[1205] be lasht:
And, for they could no crime agaynst him fynd,
They forgde a fault the people’s eyes to blinde,
And told he should by sorceries pretend
To bring the king vnto a spedy ende.

47.

Of all which poynts he was as innocent

As is the babe that lacketh kindely breth:
And yet condemned by the king’s assent,
Most cruelly put to a shamefull death:
This fierd my hart, as foulder doth the heath:
So that I could not but exclame and cry,
Agaynst so great and open iniury.

48.

For this I was commaunded to the tower,

The king my brother was so cruel harted,
And when my brother Richard saw the hower
Was come, for which his hart so sore had smarted,
He thought it best take time before it parted:
For he endeuoured to attayne the crowne,
From which my life must nedes haue held him downe.

49.

For though the king within a while had died,

As nedes he must, he surfayted so oft,
I must haue had his children in my guyde,
So Richard should besyde the crowne haue coft:
This made him ply the while the wax was soft,
To finde a meane to bring me to an ende,
For realm-rape spareth neyther kin nor frend.

50.

And whan hee sawe how reason can asswage

Through length of time my brother Edward’s ire,
With forged tales hee set him newe in rage,
Till at the last they did my death conspire:
And though my truth sore troubled their desire,
For all the world did knowe mine innocence,
Yet they agreede to charge mee with offence.

51.

And, couertly, within the tower they calde

A quest, to geue such verdite as they should:
Who what with feare and what with fauour thralde,
Durst not pronounce but as my brethren would:
And though my false accusers neuer could
Proue ought they sayd, I guiltlesse was condemned:
Such verdites passe where iustice is contemned.

52.
This feate atchiued, yet could they not for shame
Cause mee bee kild by any[1206] common way,
But like a wolfe the tyrant Richard came,
(My brother, nay my butcher I may say)[1207]
Unto the tower when all men were away,[1208]
Saue such as were prouided for the feate:
Who in this wise did straungely mee entreate.

53.

His purpose was with a prepared string

To strangle mee: but I bestird mee so,
That by no force they could mee therto bring,
Which caused him that purpose to forgo:
Howbeit they bound mee, whether I would or no,
And in a but of malmesey standing by,
Newe christned mee, because I should not cry.[1209]

54.

Thus drownde I was, yet for no due desert,

Except the zeale of justice bee a crime:
False prophecies bewitcht king Edward’s hart,
My brother Richard to the crowne would clime:
Note these three causes in thy rufull rime,
And boldly say they did procure my fall,
And death of deaths most straunge and hard of all.

55.

And warne all[1210] princes prophecyes to eschue,

That are to darke and doubtfull to be knowen:
What God hath sayd, that cannot but ensue,
Though all the worlde would haue it ouerthrowne:
When men suppose by fetches of theyr owne
To fly[1211] theyr fate, they furder on the same,
Like quenching blastes which[1212] oft reuiue the flame.
 56.

Will princes therefore, not to thinke by murder

They may auoyde what prophecyes behight,
But by theyr meanes, theyr mischiefes they may furder,
And cause God’s vengeaunce heauier to alight:
Woe worth the wretch that striues with God’s foresight:
They are not wise, but wickedly do arre,
Which thinke yll deedes due destenies may barre.

57.

For if wee thinke that prophecyes be true,

We must beleue it cannot but betyde,
Which God in them foresheweth shall ensue,
For his decrees vnchaunged doe abide:
Which to be true my brethren both haue tryed,
Whose wicked warkes warne princes to detest,
That other’s harmes may keepe them better blest.[1213]
 [By that this tragedy was ended, night was so nere come that
wee could not conueniently tary together any longer: and therefore
sayd maister Ferrers: “It is best my maisters to stay here. For wee be
come now[1214] to the end of Edward the fourth’s raigne.[1215] For
the last whom wee finde vnfortunate therein, was the duke of
Clarence: in whose behalfe I commend much that which hath bene
noted. Let vs therefore for this time leaue with him, and this day
seauen nights hence, if your busines will so suffer, let vs all meete
here together[1216] agayne. And you shall see that in the meane
season I will not only deuise vpon this my selfe, but cause diuers
other of my acquayntance, which can doe very well, to helpe vs
forwarde with the rest.” To this euery man gladly agreed. “Howbeit,”
sayd[1217] another, “seing we shall end at Edward the fourth’s end,
let himselfe make an ende of our daye’s labour, with the same
oration which maister Skelton made in his name, the tenour whereof,
so far as I remember, is as foloweth.”][1218]
 Howe King Edward the fourth[1219]
through his surfeting and
vntemperate life, sodaynly dyed in the
middest of his prosperity, the nynth of
Aprill, Anno 1483.
1.

Miseremini mei yee that bee my frendes,

This world hath formed mee downe to fall:
How may I endure whan that euery thing ends?
What creature is borne to be eternall?
Now there is no more but pray for mee all,
Thus say I, Edward, that late was your king,
And twenty-two[1220] yeares ruled this imperiall,[1221]
Some vnto pleasure and some to no lyking:
Mercy I aske of my misdoyng,
What avayleth it frendes to bee my foe?
Sith I cannot resist, nor amend your complayning,
Quia ecce[1222] nunc in puluere dormio.

2.

I sleepe now in mould as it is naturall,

As earth vnto earth hath his reuerture:
What ordayned God to bee terrestriall,[1223]
Without recourse to the earth by nature?
Who to liue euer may himselfe assure?
What is it to trust to mutability?
Sith that in this worlde nothing may endure:
(For now am I gone that was late in prosperity)
To presume thereuppon it is but[1224] vanity:
Not certayne, but as a chery fayre full of wo:
Raigned not I of late in great prosperity?[1225]
Et ecce in nunc puluere dormio.

3.

Where was in my life such an one as I,

While lady fortune had with me[1226] continuaunce:
Graunted not shee mee to haue victory,
In England to raigne and to contribute Fraunce?
Shee tooke mee by the hand and led me a daunce,
And with her sugred lips on mee shee smyled,
But what for dissembled countenaunce,
I could not beware till I was beguyled:
Now from this world shee hath mee exiled,
Whan I was lothest hence for to goe,
And am in age as[1227] (who sayth) but a childe,
Et ecce nunc in puluere dormio.

4.

I had enough, I held mee not content,

Without remembraunce that I should dye:
And moreouer to encroch redy was I bent,
I knew not how long I should it occupye,
I made the towre strong, I wist not why:
I knew not to whom I purchased Tartersall:
I mended Douer on the mountayne hye:
And London I prouoked to fortify the wall:
I made Notingham a place full royall:
Windsore, Eltam, and many other mo,
Yet at the last I went from them all,
Et ecce nunc in puluere dormio.

5.
Where is now my conquest and victory?
Where is my riches and royall array?
Where be my coursers and my horses hye,
Where is my myrth, my solace, and my play?
As vanity to nought all[1228] is wythered away:
O lady Bes long for mee may you call,
For I am departed vntill dome’s day:
But loue you that lord that is soueraine of all:
Where bee my castles and buildings royall?
But Windsore alone now haue I no moe,
And of Eton the prayers perpetuall,
Et ecce nunc in puluere dormio.

6.

Why should a man bee prowde or presume hye?

Saint Bernard thereof nobly doth treate,
Saying a man is but a sacke of stercory,
And shall retourne vnto wormes meate:
Why, what became of Alexander the great?
Or else of strong Sampson, who can tell?
Were not wormes ordaynde theyr flesh to freate?
And of Salomon that was of wit the well,
Absolon preferred his hayre for to sell,
Yet for his bewty wormes eate him also,
And I but late in honoures did excell,
Et ecce nunc in puluere dormio.

7.

I haue played my pageant, now am I past,

Yee wot well all I was of no great elde:
Thus all thing concluded shalbe at the last,
When death approcheth then lost is the fielde:
Then seing this world me no longer vpheld,
(For nought would conserue mee here in this place)
In manus tuas Domine my spirit vp I yeelde,
Humbly beseeching thee, O God, of thy grace,
O you courteous commons your heartes embrace,
Beningly now to pray for mee also,
For right well you[1229] know your king I was:
Et ecce nunc in puluere dormio.[1230]
 [Whan this was sayd, euery man for that[1231] time tooke his
leaue of other, and departed (for then it waxed darke) appointing a
new day of meeting, which being come, we met all together againe.
And whan we had saluted one another, then one tooke the booke,
and began to read the story of king Edward the fifte: (for there wee
left) and when hee came to the apprehending of the lord Riuers:
“Stay there I pray you,” sayd I, “for here is his complaint. For the
better vnderstanding whereof, you must imagine that he was
accompanied with the lord Richard Gray, Hawt, and Clappam,
whose infortunes hee bewaileth after this maner.”]