Clinical E The Journal ofmbryology … · combines the beneﬁ ts of a precurved guiding catheter...

Volume 13, Issue 2 • Summer 2010 ISSN 1941-1901

The Journal of

Clinical EmbryologyPRSRT STDUS POSTAGE

PAIDPERMIT 3933

SO FLA FL 330

The Egg, just prior to ovulation, surrounded by nutrient cells like a radiant wreath, corona radiata © Lennart Nilsson/SCANPIX

The Official Journal ofThe College of Reproductive Biology - American Association of Bioanalysts.

w w w.embr yologists .com

Cook Medical now offers you even more choices in embryo transfer. The Guardia™ AccessET combines the benefi ts of a precurved guiding catheter with the advantage of EchoTip® technology. The echogenic tip allows the catheter to be seen more clearly under ultrasound, which means that embryos can be transferred with greater accuracy.

The Guardia AccessET also features Microvol™ technology, which decreases the volume of media required for transfer, reducing the possibility of embryo migration and optimizing chances for successful implantation.

For more information, visit CookARTlab.com or contact your Cook Medical representative.

Cook Medical—Simplifying procedures for patients and clinicians.

Improve uterine accessand embryo placement.

Guardia™ AccessETC U R V E D E M B RYO T R A N S F E R C ATH E T E R ©

CO

OK

201

0

WH

-BA

DV

-JC

EGA

E-EN

-201

004

EM

BRYO TRANSFER

CookARTlab.com

AORTICINTERVENTION

CRITICALCARE

ENDOSCOPYINTERVENTIONALRADIOLOGY

LEADMANAGEMENT

PERIPHERALINTERVENTION

SURGERY UROLOGYWOMEN’SHEALTH

WH-BADV-JCEGAE-EN-201004.indd 1 4/16/10 3:32 PM

The Journal of Clinical Embryology™ Volume 13, Issue 2

3

Editor’s Corner: “EmbryoSpeak”

Speaking from the Summit Ken Drury, Ph.D., Editor

TABLE OF CONTENTS

Editor’s Corner - “Speaking from the Summit” ... 3Ken Drury, Ph.D., Editor

Clinical Embryologist’s Summit Conference: A Historical Perspective ................................5Dean E. Morbeck, Ph.D., HCLD (ABB)Jeffrey M. Jones, Ph.D., HCLD (ABB)

Back to Basics: pH for the ARTisan (Importance of pH and Buffer Selection in IVF)..........9Jason E. Swain, PhD

Techniques to achieve low oxygen tension for culture of human embryos .....................29Michael L. Reed, Ph.D. HCLD (ABB)

MicroSecure Vitrification (µS-VTF) Procedure: Optimum simplicity, security, cost-savings and effectiveness combining FDA-approved products ......33Mitchel C. Schiewe, PhD

Supplementation of freeze and thaw solutions with a globulin-rich protein source improves post-thaw survival and implantation of control-rate cryopreserved blastocysts ....41Joseph M. Kramer, M.Sc.

Opinions expressed in each article are solely those of each signatory of that article and so may not or do not reflect the opinions of any unsigned Editorial Board member nor -- unless he is an explicit signatory -- of the Editor and/or the Publisher.

For the 11th year in a row, Madison Wisconsin was a hot bed of intrigue as Clinical Embryologists converged on this tranquil college town to take part in a high level “Summit” meeting to discuss the

searing issues confronting those who labor within ART Laboratories (see Clinical Embryologist’s Summit Conference: A historical perspective by Drs. Morbeck and Jones p5).

It may be of interest, for those not in attendance, to know exactly what transpired during this “Summit” meeting. Just hearing rumors of what may have taken place in the side corridors during much needed breaks does not reveal the importance of the issues discussed. It is therefore imperative to obtain direct access to the presentations that set the course of events for those in attendance.

But, before these subjects are dissected in more detail, it is important to understand why such high-level focused meetings are necessary. ART laboratory specialists are regularly confronted with new ideas and updated protocols in order to successfully grapple with the ever increasing complexities of the new technologies being harnessed in ART laboratories in an effort to successfully treat any form of infertility. Now, it is certainly acknowledged that super mega-meetings have vast advantages when it comes to circulating newsbites and press releases. However, they can easily spur the imagination to total excess and, in the end, only the safe and familiar confines of the ART laboratory are able to restore solace and clarity.

This is in stark contrast to the more collegial and relaxed atmosphere of those smaller focused meetings such as the “Summit’. Attendees immediately feel the freedom of involvement and, in this environment of camaraderie, new issues can be brought into focus more rapidly in an effort to bore down to the core of actual understanding. This core understanding greatly enhances the prospects for new technologies to be successfully implemented into clinical practice or old ones to be adapted to more practical functions. It also enables individuals to return to their own programs with a renewed sense of purpose and confidence, as well as, giving them more contacts with other lab personnel who speak embryologese.

Of course, the “Summit” is not the only such gathering available to clinical embryologists. Some are sponsored by national organizations such as the American Association of Bioanalysts and the College of Reproductive Biology (AAB/CRB), others are more regional such as the New England Fertility Society (NEFS), The Midwest Reproductive Symposium (MRS); Northern California Association of Reproductive Biologists (NCARB); Southeastern Embryology Society; Pacific Coast Reproductive Society (PCRS), and the Florida Society of Reproductive Endocrinology and Infertility-FSREI (embryologists and nurses included).


4

THE JOURNAL OF CLINICAL EMBRYOLOGY™MISSION STATEMENT:

The Journal of Clinical Embryology™ is committed to reporting significant,

accurate and up-to-date scientific articles and information concerning issues of importance to clinical laboratory embryologists, andrologists

and those professionals engaged in the science of human assisted reproductive technology (ART)

and infertility medicine.

MEDICAL MARkETS CONSULTANTS, INC.

755 8th Court, Suite #4PO Box 650790

Vero Beach, FL 32965-0790Phone 352.331.5235

Fax 352.331.6250 E-mail: [email protected]

www.embryologists.com

New Subscription Rate Details for the Journal of Clinical Embryology (JCE)

Dear Subscribers,

The Journal of Clinical Embryology has been considered the primary journal covering all aspects of clinical ART laboratory interests for many years, and during this time, our subscriber base has increased considerably. However, and unfortunately, the costs of bringing you our Journal free of charge have now risen to the point that we are required to initiate a subscription fee for all our subscribers. We feel confident that our readers will want to support and maintain access to the JCE so that a minimal fee for continued service will be readily accepted.

In light of the current economic realities, and projecting our growth into the future, these changes will give the JCE the opportunity to grow and expand along with the continued maturation of our clinical profession and the College of Reproductive Biology / American Association of Bioanalysts.

Subscription fees will become effective January 2010 and reflect the subscriber’s rights to four issues of our hardcopy JCE as well as continued access to the JCE website, “www.embryologists.com”.

Additional details can be found on the subscription page within this issue of the Journal, as well as, on the JCE website, www.embryologists.com. Very Sincerely Yours, Fred Zander, Publisher

These meetings all play an important role in the interweaving of knowledge amongst those who are responsible for the introduction of that knowledge into clinical use. They also underscore the increasingly important role of providing an understanding, and appreciation of, the role that each ART laboratory personnel plays in bringing about successful outcomes for our infertility patients.

This issue of the Journal now allows you to listen in on what transpired during the latest “Summit Meeting”. Articles by Drs. Swain, Reed and Scheiwe present cutting edge topics which make for great reading and inspired discussions amongst embryologists of all ages.

Also, read about how a young graduate student makes his way into the world of ART in “Supplementation of freeze and thaw solutions with a globulin-rich protein source improves post-thaw survival and implantation of control-rate cryopreserved blastocysts” on page 41 by Joseph M. Kramer, M.Sc.

Ed Notes: The JCE would like to acknowledge and thank Drs. Swain, Reed and Scheiwe for making their manuscripts available for publication. n


5

Dean E. Morbeck, Ph.D., HCLD(ABB)Mayo Clinic

[email protected]

Jeffrey M. Jones, Ph.D., HCLD(ABB)WiCell Research Institute

[email protected]

Clinical Embryologist’s Summit Conference:A Historical Perspective

The Clinical Embryologist’s Summit Conference is an annual event hosted by the University of Wisconsin-Madison that is designed to

provide participants with an informal setting to share ideas and interact with invited speakers and other embryologists. As indicated by its name, this meeting is truly a “Summit” during which all attendees are expected to participate in open and honest discussion on a specific topic of common interest. As such, this conference is unique among scientific meetings.

The genesis of the Summit occurred about 10 years ago during a time of dynamic change in clinical embryology. At the turn of the century, blastocyst culture and PGD were being adopted by many clinics

and this was soon followed by advances in vitrification and oocyte freezing. The FDA and its plethora of regulations was next to appear on the landscape. These topics were presented at most scientific meetings in a traditional format: a series of invited speakers lectured to an audience for a specific period of time which was immediately followed by a limited time for questions. While this paradigm provided an efficient mechanism to present new scientific data, the exchange of information was unidirectional. Discussions on e-mail listservers and conferences hosted on the internet allowed additional exchanges among embryologists, but the exchanges were restricted by time zone differences and required the management of discussion mediators.

Year Topic Summit Organizer2000 Blastocyst Culture J. Jones

2001 Pre-implantation Genetic Screening P. Andresen

2002 Infectious Disease Testing (FDA Regulation) A. Thornhill

2003 QA/ QC Assays D. Morbeck

2004 Cryopreservation A. Sparks

2005 Ethics and ART D. Olive

2006 Vitrification M. Dow

2007 Embryo Screening D. Morbeck

2008 Current Topics W. Megid & D. Morbeck

2009 Fertility Preservation C. Bormann

2010 Improving IVF Outcomes from the Laboratory J. Swain

The American Board of Bioanalysis (ABB), an internationally recognized certifying board established in 1968, evaluates and identifies, on a nondiscriminatory basis, individuals who meet ABB’s minimum requirements of competency and who can establish their competency to perform as clinical laboratory directors, consultants and supervisors. ABB certification is available by examination in these disciplines: Andrology, Embryology, Molecular Diagnostics, Chemistry, Microbiology, Hematology, Diagnostic Immunology and Public Health Microbiology.

For more information, contact: ABB, 906 Olive Street, Suite 1200, Saint Louis, MO 63101-1448, phone: 314-241-1445, fax: 314-241-1449,

email: [email protected], web: www.abbcert.org.

ABB certification is available for: High-complexity Clinical Laboratory Director (HCLD) - A lab director who has expertise in high complexity testing in at least one clinical lab discipline or specialty. Embryology Laboratory Director (ELD) - A lab director who has expertise in the specialty of assisted reproductive technology (ART) laboratory procedures in an embryology laboratory. Consultant - A clinical consultant provides consultation to the clients of a lab performing moderate or high complexity testing regarding the appropriateness of the testing ordered and interpretation of test results. Technical Supervisor (TS) - A technical supervisor is responsible for the technical and scientific oversight of a lab performing high complexity testing. General Supervisor (GS) - A general supervisor is responsible for the day-to-day supervision or oversight of lab testing operations and personnel performing high complexity testing.

ABB Examination Dates

Medical Technologists working in assisted reproductive technology laboratories may qualify for the AAB Board of Registry Medical Technologist certification [MT(AAB)] in these disciplines:Andrology (Male Infertility, Semen Analysis, Sperm Function Tests, Sperm-Cervical Mucous Interaction, Anti-Sperm Antibodies), and Embryology (Oocyte Identification and Fertilization, Micromanipulation, Embryo Culture, and Cryopreservation.

The CLIA ’88 regulations recognize ABB as a certifying agency for laboratory directors and clinical consultants. Most state laboratory regulatory programs also recognize ABB’s director certification.

Apply online at www.abbcert.org. Processing of ABB applications for board review requires six to eight weeks. Avoid delays in processing by completing the application in its entirety and forwarding to ABB along with required documentation.

ABB is an internationally recognized, CLIA-approved certifying board

Board Certification in Andrology and Embryology

June 5, 2010Public Health Microbiology & General Knowledge ONLYCincinnati, Ohio Prior to the Association of Public Health Laboratories (APHL) Annual Meeting

October 22, 2010Denver, Colorado Prior to the American Society for Reproductive Medicine (ASRM) Meeting

May 12, 2011Hyatt Regency Austin Austin, Texas

Embryologists and Andrologists:

Get Your Technologist Certification, MT(AAB)

You can qualify for MT(AAB) certifica-tion with a Bachelors or Associate De-gree with a major in Biology, Chemistry, Biochemistry, or Medical Technology.*

Examination Dates

Thursday, June 17, 2010Indian River State College, Fort Pierce, Florida Monday, August 23, 2010Cedars-Sinai, Los Angeles, California and Washington Hospital, Fremont, California Friday, September 24, 2010Miami-Dade College, Miami, FloridaFriday, December 3, 2010Sheridan Technical Center, Hollywood, Florida

* Some experience is required in addition to passing the examination to become fully certified.** At this time, only Florida and Georgia license MTs in the disciplines of Andrology and Embryology. A Bachelors Degree is required in Georgia.

The AAB Board of Registry is an internationally recognized, CLIA-approved certifying board. Apply online or obtain more information at www.aab.org or contact the AAB Board of Registry, email: [email protected], 906 Olive Street, Suite 1200, St. Louis, MO 63101-1448, phone: 314-241-1445, fax: 314-241-1449.

MT(AAB) Exams are Approved for Florida and Georgia Licensure **


7

Jeff Jones recognized that the time had come to gather embryologists and provide them with an environment for open exchange of information. Thus the inaugural Clinical Embryologist’s Summit Conference was held in Madison immediately preceding the annual Society for the Study of Reproduction meeting hosted by the University of Wisconsin in 2000.

In contrast to traditional meetings, The Clinical Embryologist’s Summit Conference is designed to allow attendees to have control of what is presented and discussed. The intent is to create an engaging free-for-all: the invited speaker should NOT be able to complete their entire presentation in the time allotted! With this new paradigm, participants continually ask questions of both the invited speaker and other attendees. Attendees are encouraged to present confirmatory or conflicting data, share their own experiences and insights and to openly express their opinions. By being directly engaged and

truly responsible for the success of the conference, the attendees come away with a much improved understanding and appreciation of the information discussed.

The Clinical Embryologist’s Summit Conference has been held for 10 consecutive years and has demonstrated that this new paradigm can be successful. The past 10 Summit Conferences have focused on the topics shown in the chart on page 5.

This year’s Clinical Embryologist’s Summit Conference (Improving IVF Outcomes from the Laboratory) was organized by Jason Swain and was held on Saturday, May 1st. The Journal of Clinical Embryology has graciously allowed all of the 2010 invited speakers to publish their presentations in this and the following issue of the journal. We hope that this tradition and Clinical Embryologist’s Summit Conference will continue for at least another 10 years. n

I WANT MY JCE!I WANT MY JCE! I WANT MY JCE!

(FOR THOSE WHO HAVE FORGOTTEN TO PAY FOR

THEIR SUBSCIPTIONS!)

THE RESULTS ARE IN.

YOU HAVE A STORAGE PROBLEM.

www.reprot.com

Sooner or later, every growing IVF Center

will face this problem. We’ve seen it coming for

years. So we’ve been refining a shipping and

storage protocol to answer this rapidly

growing need. It’s a turnkey solution that

removes the risk and worry from your

office, and yet, assures that your patient’s

specimens are readily available when you

need them. And best of all it costs your clinic

nothing. Call us today to assure your center is

prepared to handle the risk of potentially

infectious tissue storage.

Mpls/St. Paul, Minnesota • 1-888-489-8944 | Fort Lauderdale, Florida • 1-888-953-9669Reno, Nevada • 1-888-831-2765


9

Back to Basics: pH for the ARTisan(Importance of pH and Buffer Selection in IVF)

Expanded from a presentation for the 11th Clinical Embryologist’s Summit Conference (Improving IVF Outcomes from the Laboratory)

Madison WI, May 1, 2010

Jason E. Swain, PhD University of Michigan

Center for Reproductive [email protected]

As embryologists, we strive to optimize culture conditions and improve embryo development by minimizing stresses imposed on gametes and embryos during their manipulations within the in vitro environment. We accomplish this through precise handling of cells and supplies within the lab, as well as tedious attention to detail. Indeed, the devil is in the details when it comes to improving culture conditions. It is readily apparent that improper set-points in growth conditions are stressors negatively impacting gametes and embryos, whether they be improper media energy substrate composition, temperature, or osmolality. However, periodic fluctuations in environmental conditions are also harmful stressors, as these are easily transduced into deleterious intracellular perturbations. One such environmental parameter, which not only requires that strict attention be paid to its set-point and that is also especially susceptible to these damaging oscillations, is pH.

pH DefinedThe topic of pH has been reviewed nicely in a

previous issue of this publication (Pool 2004). To quickly summarize, pH is a measure of the acidity or alkalinity of a substance, where acids are defined as substances that increase H+ ion concentration and bases are things that decrease this concentration. Thus, pH is really the measure of H+ ion concentration. Though, perhaps more importantly, pH is dynamic. The measure depends on the association/disassociation of compounds in solution and any factors which influence this balance, such as temperature. Thus, pH can change and it can do so rapidly; a point that has dramatic implications for IVF.

From a practical standpoint, it may be helpful to view pH as occurring in 3 phases within the laboratory: equilibration, set-point, and stabilization (figure 1).

These 3 phases all play important roles in minimizing cellular stress, and each stage needs to be considered for optimizing laboratory conditions.

Figure 1. Three phases of pH experienced during in vitro culture.


10

pH RegulationExtracellular pHIf pH is so sensitive and important, it would be

beneficial to know how we both set and regulate the extracellular pH of our culture media (pHe). Traditionally, pHe is primarily the result of a balance between levels of CO2 in the cell culture incubator and the amount of bicarbonate in the media. Gaseous CO2 dissolves in solution to produce carbonic acid, which reaches equilibrium with the amount of dissolved bicarbonate. Generally the bicarbonate level, supplied as sodium bicarbonate, is set by the media manufacturing company. Thus, to regulate the set-point of our media pHe, we (the embryologist) control the CO2 value on the incubator. This is an inverse relationship, with pHe decreasing as CO2 levels increase.

This understanding of pHe sheds light on the 3 phases of pH (figure 1). Equilibration timing then depends on diffusion of CO2 into the media and the timing of the above reaction. Thus, volume of media, surface area, use of oil overlay, and even the type of lid/dishware can influence this gas exchange and equilibration timing. For the set-point of media pH, though CO2 and bicarbonate are the major contributors to pH, they are not the only elements to consider. As an example, protein source and concentration can both affect pHe. Additionally, elevation of the laboratory may also be a factor as air pressure changes with altitude. Thus, the same basal media may not yield the same equilibration time or set-point pHe from laboratory to laboratory, even if the same CO2 levels are supplied. We begin to now see the problem with simply setting a specific CO2 concentration on our incubators. As we’ll discuss later, the inappropriateness of this practice is further demonstrated by the fact that incubator CO2 readings are not always accurate (figure 5). Regarding stabilization of pH, monitoring patient/incubator ratios to minimize door openings/closings, use of inner doors, and oil overlay help reduce gas exchange that perturb CO2 levels can lead to pHe fluctuation. Additionally, use of smaller incubators can aid in gas recovery.

Intracellular pHRegulation of intracellular pH (pHi) is an important

cellular function necessary to maintain intracellular homeostasis. Cells contain various mechanisms to regulate pHi that are activated at various pH values,

though specific mechanisms will vary from cell type to cell type (figure 2). Short-term regulation of pHi is achieved by the limited physiochemical buffering capacity of the cytoplasm and proteins (Lane and Gardner 2000). Common regulatory systems to combat intracellular acidosis include the sodium dependent HCO3-/Cl- exchanger (HCE) and the Na+/H+ antiporter, which adds or removes bicarbonate and hydrogen ions from within the cell, respectively. To combat alkalosis, cells may contain the HCO3-/Cl -exchanger (figure 2). Additionally, though not a conventional pHi regulator per se, it should be mentioned that the monocarboxylate co-transporter (MCT) can also influence pHi (Gibb et al. 1997, (Herubel et al. 2002).

Figure 2. Mechanisms regulating internal pH of embryos (pHi) and the set points at which they activate in human embryos (Phillips et al. 2000).

Studies show that embryos from a variety of species possess active pHi regulatory mechanisms, (Zhao et al. 1995; Zhao and Baltz 1996; Barr et al. 1998; Dale et al. 1998; Edwards et al. 1998; Edwards et al. 1998; Lane et al. 1998; Lane et al. 1999; Lane et al. 1999; Lane and Bavister 1999; Phillips et al. 2000; Phillips et al. 2002). Interestingly, the pHi value has been repeatedly shown to be approximately 7.1, which is much lower than the 7.4 of blood (Table 1a). This fact alone has implications on culture practices of embryos. In human embryos,

Zander Scientific, Inc.772-569-5955 • www.zandair.com

An independent report prepared by Alpha Environmental, Inc. on the efficiency of the ZAND-AIR PCOC3™ is now available.

Technology Update Don’t underestimate the importance of… Air Purity

(Destroying VOCs in IVF Laboratories)

“As I travel to IVF labs throughout the world, I visit with physicians and Lab Directors that are realizing pregnancy rates in the 65% range in young patients…all of them understand the absolute importance of purity in their lab air”….. F. Zander

The purity of ambient air is a process that many industries throughout the world continue to focus on improving. Much of the research and development is centered on removing both dry and liquid particles. In the industrial sector continued improvements have been made to comply with EPA regulations, and reduce particulates to improve general working conditions.

The Destruction of Microbes and VOCsIn IVF laboratories, physicians’ offices, hospitals, and clinics, it is well documented that ambient air carries harmful VOCs (volatile organic compounds). Styrenes, formaldehydes, glutealdehydes, toluene, as well as microbes (bacteria, viruses, mold and fungi), are all present in our laboratories. Perfumes, lipstick, deodorants, as well as ‘any smell’ from the outside environment are potential molecules that deter the development of embryos. At a molecular level these compounds are carried in the circulated air.

In the IVF laboratory there is only one way to solve this issue. That is to destroy the harmful molecules. There is no filter that destroys molecular structures. HEPA and activated charcoal filters, while effective, destroy nothing. Most molecules are not filtered out of the atmosphere, and yet it is the molecule that in fact deters embryo growth.

The recognized technology that does destroy VOCs and microbes is photocatalytic oxidation (PCO). TiO2 energized by 257.3 nm UVC light activates the photocatalytic oxidation action, creating free radicals that in turn destroy both molecules and cellular structure.

The Ultimate Medical Laboratory Purification System

ZAND-AIR PCOC3™Zander uses a patented process to affix TiO2 to a solid substrate so it interacts with UVC radiation at 253.7 nm. This wave length is not adsorbed by the oxygen molecules, and therefore does not create any ozone - O3 (trioxygen). Naturally occurring ozone in the atmosphere is converted by the TiO2 / UVC interaction into normal dioxygen (O2), or other ambient gases. These gases are then converted to benign matter, including diatomic oxygen (O2), which forms 20.9% of our ambient atmosphere.

While the photocatalytic process is very fast, the number of individual photocatalytic actions occurring in the PCO chamber are multiple and very complex. The efficiency of this process is therefore increased by placing three such PCO chambers sequentially in a single frame, thus increasing the ‘dwell time’ of the airstream through the ZAND-AIR PCOC3™ equipment.

Placing several PCOC™ units sequentially increases the dwell time for the UV light interaction with the photocatalyst, thus multiplying the decomposition of VOCs and pathogens.

GERMICIDAL ULTRAVIOLET LIGHT kills disease causing airborne viruses and bacteria on contact.

ZAND-AIR PCOC3™ zIVF-AIRe™ 100C

VIsIT

us A

T EsH

RE

Rome • Ju

ne 27-3

0

BooTH

#A2

Subscriber Details:Subscriber Name: __________________________________________________________Subscriber Email: __________________________________________________________Company/Institute: ________________________ Phone Number: ___________________

Delivery Address:Line 1: _______________________________________________________Line 2: _______________________________________________________Line 3: _______________________________________________________Postal Code: ____________________City/Town: ______________________Province/State __________________Country: ________________________

Credit Card Payment and Authorization:Select a payment Method: MASTERCARD VISA AMERICAN EXPRESSAmount to be charged to your card: $______________________Credit Card number: ________ ________ ________ ________CVN or CVV2 Number: ________ Expiration Date: Month ______ Year ______Name as it appears on Credit Card: ____________________________________________

Please Note:I/we hereby authorize MMC to process payment requests for goods and services which we have ordered and purchased from MMC, by debiting the above mentioned card number without showing the card.

We are aware that we can cancel this authorization at any time by giving notice in writing to MMC. Individual signing shall be authorized to sign on behalf of Purchaser / Credit Card Owner.

FoRM

suBsCRIBING...

Subscription

Thank you for

PLEASE FAX COMPLETED FORM TO US+1-772-569-9667

DIRECTIoNs:Effective Jan 1, 2010 a yearly subscription fee is required for both receiving the mailed hard copy of the Journal of Clinical Embryology, as well as for full access in detail to the website www.embryologists.com. Please arrange for your yearly payment in order to continue receiving both your copy of the Journal of Clinical Embryology and have complete access to our website. Please fax this completed form to us. We will mail you a receipt for your subscription.United States ........................................US $25.00 Mexico and Canada..............................US $30.00 Outside North America .........................US $40.00Worldwide: Access to Website Only .....US $20.00

Simply fi ll out this form in its entirety and fax to our offi ce. +1-772-569-9667 or +1-772-569-4430

Medical Market Consultants, Inc.Tel: +1-352-331-5235 Fax: +1-772-569-9667 Email: [email protected] Web site: www.embryologists.com

Clinical EmbryologyTh e Journal of


13

though the pHi of the embryo initially follows the pHe, the HCO3-/Cl- exchanger activates when pHi rises above 7.2-7.3 (Phillips et al. 2000). To combat acidosis, the Na+/H+ antiporter activates when pHi drops below 6.8, and the Na+ dependent HCO3-/Cl- exchanger operates <7.0 (Phillips et al. 2000) (Table 1b) Furthermore, morula and blastocyst stage embryos appear to have more rigorous control over their pHi, possibly due to formation of tight-junctions between cells (Edwards et al. 1998). This demonstrates the plasticity of embryo development, and explains their ability to grow over a variety of pHe conditions. Physiologically, this makes sense, as the in vivo developing embryo appears to adapt to the differing pHe environment of the alkaline oviduct and more acidic uterus (Elrod and Butler 1993; Kane et al. 2002; Hugentobler et al. 2004). However, as discussed below, just because embryos can form blastocysts over a range of pHe conditions, it does not mean that resulting embryo quality is equivalent.

Table 1 . A) Internal pH (pHi) of human oocytes and embryos (adapted from Phillips et al. 2000). B) Activation points of various pHi regulatory mechanisms.

Though embryos possess functioning pHi regulatory mechanisms, oocyte pHi regulation is a paradoxical event (Fitzharris and Baltz 2009). Growing mouse oocytes in the follicle lack pHi regulatory capacity (Erdogan et al. 2005). While fully-grown immature mouse oocytes can regulate pHi by using the HCO3

-/Cl- exchanger, these mechanism are inactivated during meiotic progression (Phillips et al. 2002). As a result, denuded mature MII oocytes are incapable of actively regulating pHi. This results in a sensitive cell stage, susceptible to even slight deviations in pHe. Interestingly, surrounding cumulus cells convey pHi regulatory capacity to the enclosed oocyte through gap junctions (Fitzharris and Baltz 2006; FitzHarris et al. 2007), a factor which has implications in clinical IVF procedures, such as ICSI, in which cumulus cells are removed. Interestingly, the bovine and human oocyte appear to have very limited ability to combat alkalosis (Dale et al. 1998; Lane and Bavister 1999; Phillips et al. 2000). Therefore, there may be slight species variations in oocyte pHi regulation, or regulatory capacity may be impacted by in vitro culture conditions (Lane and Gardner 2000). Continued work is required to determine exactly how and why oocyte pHi regulatory capacity is turned-off during maturation and re-initiated following fertilization. This information will prove valuable in the continued improvement of culture conditions for emerging technologies such as clinical IVM.

Physiologic Importance of pHEmbryosNow that a basic ground work for defining and

regulating pH has been established, the tremendous impact it has on cells should be discussed. pH controls several intracellular processes that can impact embryo development. As an example, raising (~7.4) or lowering (~6.8) pHi in mouse embryos for only 3 hours disrupts localization of mitochondrial and actin microfilaments compared to controls (~7.2) (Squirrell et al. 2001). Even minor rises in pH can also dramatically impact embryo metabolism through regulation of various enzymes, such as phosphofructokinase (PFK). Raising pHi ~0.1-0.15

units significantly increased embryo glycolysis and lowered oxidative metabolism (Edwards et al. 1998; Lane et al. 2000), which can dramatically impact developmental competence. Importantly, other common practices in


14

IVF, such as vitrification of embryos, reduces the ability of these already sensitive cells to regulate pHi for about 6 hours (Lane et al. 2000). How different cryopreservation strategies impact pH regulation remains unknown. Finally, more recent research suggests that pHe can not only affect embryo development, but resulting fetal development as well. Lowering the pHi of 1-cell mouse embryos from 7.25 to 7.1 for 19h resulted in significantly fewer blastocyst cell numbers, higher levels of apoptosis and reduced fetal size/weight compared to controls (Zander-Fox et al. 2008).

OocytesAs mentioned, denuded mature oocytes lack, or have

diminished ability to regulate pHi and are therefore dependent upon pHe. Thus, in procedures such as ICSI, where the protective cumulus cells are purposefully removed, cells are created that are extremely susceptible to perturbations in pHe until several hours after fertilization occurs (Lane et al. 1999; Phillips and Baltz 1999). Little work has been done examining the

effects of pHe on the mature oocyte. This should be concerning considering the prevalence of oocyte derived aneuploidy and the potential impact pH has on the meiotic spindle. pH is known to affect embryo actin cytoskeletal elements (Squirrell et al. 2001), and the oocyte cytoskeleton is responsible for positioning of the meiotic spindle (Zhu et al. 2003; Lenart et al. 2005). It is known that alkaline pH affects microtubule assembly/disassembly in bovine brain cells (Regula et al. 1981), and similar actions may be occurring with the meiotic spindle within the oocyte. Additionally, because pH can affect embryo mitochondrial localization (Squirrell et al. 2001)), the same may hold true of oocytes. This is concerning because distribution of oocyte mitochondria is correlated to developmental competence (Bavister and Squirrell 2000; Nagai et al. 2006). Even small increases in pHi perturbs embryo metabolism (Edwards et al. 1998; Lane et al. 2000), which can profoundly affect subsequent development. Oocyte metabolism is also likely affected by pH, and oocyte metabolism has been correlated with maturational status and developmental


15

competence (Krisher and Bavister 1999; Spindler et al. 2000). Additionally, similar to embryo cryopreservation, whether vitrification of oocytes compromises the ability of resulting embryos to regulate pHi is also undetermined. All these factors indicate a strict regulation of pHe when dealing with oocytes.

SpermThough the majority of focus on pH in ART has

focused on the oocyte and embryo, pH can also impact sperm function in a variety of species, including human (Hamamah and Gatti 1998). Ram and bull sperm have a resting pHi of ~6.8 (Babcock et al. 1983; Babcock and Pfeiffer 1987) and human sperm pHi has been measured from ~6.5 to ~6.9 depending on ionic conditions and method of measurement (Brook et al. 1996; Hamamah et al. 1996; Cross and Razy-Faulkner 1997), . Resting pHi of mouse sperm appears to be ~6.5, with an increase of >0.3 units during capacitation (Zeng et al. 1996). This rise in pHi precedes Ca2+ influx in bovine, ram, and mouse sperm, which influences hyperactivation (Babcock et al. 1983; Babcock and Pfeiffer 1987; Marquez and Suarez 2007; Navarro et al. 2007), possibly in response to a zona pellucida factor (Florman et al. 1989). An increase in pHi also appears to accompany the acrosome reaction in human sperm (Brook et al. 1996). Mouse sperm do possess a sperm specific Na+ dependent HCO3

+/Cl- exchanger to regulate pHi, as well as a second undefined acid-export pathway (Zeng et al. 1996). Interestingly, it has been shown that pHe influences pHi in sperm (Babcock and Pfeiffer 1987), including that of human (Hamamah et al. 1996). Importantly, varying pHe can influence sperm motility in mammals (Emmens 1947; Pholpramool and Chaturapanich 1979), including minor effect in human (Hamamah and Gatti 1998). This may be due to enzyme dependence on pH involved with a dynein-ATPase in the axeomes of sperm flagella, similar to what occurs in sea urchin (Christen et al. 1982).

Selecting pHe for CultureThe ability of embryos to regulate pHi is evidenced

by various studies that show embryos can develop over a pHe range of ~7.0-7.4 without any discernable effect on pHi or development (John and Kiessling 1988; Lane

et al. 1998), while excursions of pHe outside this range have deleterious effects on embryo developmental competence (Leclerc et al. 1994; Zhao et al. 1995; Zhao and Baltz 1996; Lane et al. 1999; Lane and Bavister 1999). However, just because blastocysts can be formed over these pHe ranges, does not indicate that resulting embryo quality is equivalent. Drifting too far away from the pHi of around 7.1 likely stresses the embryo, as more resources are required to maintain the proper pHi.. Future studies examining resulting implantation potential or molecular/genetic profiling of embryos cultured under different pHe conditions will likely reflect this. Conventional wisdom tells us that pHe should be slightly higher than pHi to help offset the acidification that occurs as a result of intracellular metabolic processes. Thus, many laboratories culture their embryos in the range of 7.2-7.4. However, this is a wide-range with >60% difference in [H+] (Figure 3).

Figure 3. Due to the logarithmic pH scale, “minor” changes in pH are actually large fluctuations in hydrogen ion concentration. Therefore, small ranges of acceptable pH should be set to maximize laboratory consistency.

Unfortunately, there is likely no “optimum” pHe, as this will vary from media to media based on its ingredients. Anecdotal observations from various laboratories suggest that perhaps culturing cleavage-stage embryos closer to pHe 7.2 may give better embryo development. However, the amount of monocarboxylic acids, such as lactate and pyruvate, in culture media


16

can lower pHi (Gibb et al. 1997; Edwards et al. 1998). Additionally, certain amino acids, such as glycine, taurine and glutamine, act as zwitterions and help in buffering pHi (Edwards et al. 1998). Thus, embryos grown in media with different amounts of these components may have different pHi, though the pHe may be the same. Along with potential species or strain-specific requirements, this likely explains variations in the literature regarding acceptable and optimal pHe (Brinster 1965; Hershlag and Feng 2001; Summers and Biggers 2003). Regardless, it would be insightful to see a properly controlled clinical trial to determine if culturing embryos at a pH closer to 7.2 offers any benefit on embryo development, implantation, or live birth, compared to culturing embryos in the same media at a pH closer to 7.4. At the moment there is no “ideal” pHe at which to culture embryos and this is reflected in the wide ranges of acceptable pHe given by various commercial media companies (Table 2 ). Furthermore, despite the growing trend, it remains unknown whether early cleavage stage embryos prefer a slightly lower pHe than later stages of embryo development (Though as mentioned, it is known that later stages of embryos, like the morula and blastocyst, can regulate their pHi more rigorously than

early cleavage stage embryos.) That being said, there are data to suggest a slightly more alkaline pHe may benefit fertilization. Dale et al (8) found higher rates of human sperm binding to empty zona pellucidae at pHe 7.5 compared to lower pHe’s. Bavister also showed increased fertilization in hamster pH 7.4 compared to lower or higher values, though bicarbonate levels were varied to adjust pH in this study . This has lead to the common practice of fertilizing oocytes in a slightly higher pH, culturing day 1-3 embryos in a slightly lower pH, and culturing day 4-6 embryos in a slightly higher pH (high-low-high paradigm).

Practically, this changing of media pH for various culture steps means that either specialized media can be used with altering bicarbonate concentrations, or separate incubators with altered CO2 can be used to achieve the differential pHe for fertilization and embryo culture. Thus, while various commercial companies list a wide range of acceptable pHe values for their media, and some labs may argue for a benefit of a slightly lower pHe, there is no argument that regardless of your final pHe, tight regulation and a narrow acceptable pHe range are critical components of a rigorous quality control program.

Website: www.embryologists.com

Please note that The Journal of Clinical Embryology™ is available online. This site will allow you to access all prior issues of the JCE as well as selected popular past articles. You will also be able to find clinical ART laboratory protocols and subscribe online or update your mailing address.

Advertising sites are immediately available and ad placement information can be obtained at [email protected] or 352-331-5235.

Visit today and save our website to your favorites.


17

Table 2: Recommended pH ranges of various commercially available media used in clinical IVF.

MIDATL ANTICDEVICES

MEDICULTMEDIA

HUMAGENPIPETS

ORIGIO supplies each step of the way. From micropipets to entire

laboratories. From media to customized equipment. We can take

care of all your ART needs. Specialized people and products over

the entire ART fi eld mean life gets a good start with ORIGIO.

ORIGIO embraces the three product brands:

MediCult Media, Humagen Pipets and MidAtlantic Devices.

MIDATL ANTICDEVICES

MEDICULTMEDIA

HUMAGENPIPETS

ORIGIO. One stop, one solution.

www.origio.com


19

IVF Handling MediaThough techniques such as oil overlay can delay pH

increase of culture media while outside the incubator, even brief periods at room atmosphere can result in pH rising above a critical value of 7.4 (Steel and Conaghan 2008; Swain and Pool 2009). Thus, use of specialized handling media to resist changes in pH, offer tremendous pH stability and logistical advantages over bicarbonate-only buffered media for procedures such as embryo transfer and ICSI. However, even brief exposure to suboptimal handling media can compromise embryos (Gardner and Lane 1996; Farrell and Bavister 1984; Escriba et al. 2001; Palasz et al. 2008). Thus, special attention should be paid to the use of these buffers.

In the past, handling media included phosphate buffered saline solutions (PBS), and some laboratories continue using this media for oocyte retrieval. However, although the pKa value of 7.2 for PBS and its buffering capacity may be adequate, PBS lacks essential components, such as bicarbonate and metabolic substrates. This inadequacy, coupled with elevated levels of phosphate, may compromise gamete and embryo metabolic activity, as well as, disrupt organelle distribution and interfere with intracellular ionic homeostasis; including intracellular pH (Barnett and Bavister 1996; Barnett et al. 1997; Lane et al. 1999). Indeed, even brief exposure to PBS as a handling media has been shown to compromise hamster and rabbit embryo development (Farrell and Bavister 1984; Escriba et al. 2001) and result in aberrant gene expression in bovine embryos when compared to other buffers (Palasz et al. 2008). Therefore, a better choice entails the use of handling media that utilize lower concentrations of bicarbonate in conjunction with synthetic organic buffers in order to maintain media pH within a desired range. (Good et al. 1966; Good and Izawa 1972; Ferguson et al. 1980). These buffers, commonly referred to as Good’s buffers, provide supplemental buffering capacity over the physiologic pH range of approximately 6.1 to 8.3. Good’s buffers, named for and derived by Norman Good and colleagues, are organic compounds derived largely from zwitterionic amino acids. However, different cell types display varying sensitivity to individual zwitterionic buffers (Ferguson et al. 1980).

Thus, determining the suitability of specific buffers for use with mammalian gametes and embryos in IVF is crucial. Two of these Good’s buffers, commonly used in commercially available handling media for ART, are 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino)- propanesulfonic acid (MOPS), are selected based on their pKa values which are indicators of their optimal buffering capacity.

Historically, HEPES (at ~21mM) has been a standard for IVF handling media. More recently, at least two commercial companies now include MOPS in their IVF handling media. However, though both buffers are efficient and laboratories using media containing these buffers yield high success rates, there may still be room for improvement when it comes to pH buffering. Though HEPES has been widely used in IVF for years, its suitability for procedures has been questioned (Iwasaki et al. 1999; Morgia et al. 2006). However, many of the conclusions drawn concerning toxicity of HEPES are often mis-cited and not fully supported by the studies performed. Early somatic cell studies citing HEPES toxicity stemmed from light exposure and interactions with riboflavin (Zigler et al. 1985; Lepe-Zuniga et al. 1987). This is not an issue in IVF, as media are void of riboflavin. Several studies actually indicate HEPES is able to support oocyte maturation (Byrd et al. 1997; Downs and Mastropolo 1997), fertilization (Bhattacharyya and Yanagimachi 1988; Behr et al. 1990; Hagen et al. 1991) and embryo culture (Mahadevan et al. 1986; Hagen et al. 1991; Ali et al. 1993; Ozawa et al. 2006) at room atmosphere. Those studies indicating lower fertilization rates in presence of HEPES are likely due to the simultaneous reduction in bicarbonate concentrations (Lee and Storey 1986). Embryo development is supported in the presence of HEPES when bicarbonate is present, but not when bicarbonate is absent (Mahadevan et al. 1986). Furthermore, when embryos are cultured at room atmosphere and compared to controls cultured in 5% CO2, differences in development cannot be attributed to HEPES alone. Elevated CO2 within the laboratory incubator is utilized by embryos for various biochemical processes as a carbon source (Graves and Biggers 1970; Quinn and Wales 1971; Quinn and Wales 1974), and is likely beneficial over culture at room atmosphere.


20

Importantly, because of its relatively recent introduction into the field of IVF, MOPS has not received as much attention as HEPES. However, use of MOPS is not without concern for unexpected cellular actions. MOPS can interfere with taurine uptake in tumour cell lines (Wersinger et al. 2001), interact with DNA in cellular preparations (Stellwagen et al. 2000), and interfere with Cl- conductance in neurons (Schmidt et al. 1996).

Possible concern with zwitterionic buffers may be concentration dependent. Very early studies indicated HEPES (as well as TES) used at 42mM for fertilization resulted in anomalies of hamster pronuclear formation, though experiments were conducted to avoid use of bicarbonate and CO2 (Bavister 1981). Only when

HEPES exceeded 35mM was any increased embryo fragmentation observed in pig embryos (Iwasaki et al. 1999). Interestingly, it has been demonstrated that under appropriate conditions, 25mM of the zwitterionic buffers HEPES, MOPS or DIPSO has no adverse effect on mouse embryo development, and that there are no adverse affects even up to 50mM when cultured with 25mM NaHCO3 in ~5% CO2, (Swain and Pool 2009; Swain and Pool 2009). Though there may be species specific sensitivities to certain buffers, and lot-to-lot variation in buffer quality that may be a concern, results demonstrate that when adequately controlled for other factors such as osmolality, ionic composition, gas levels and pH, specific zwitterionic buffers are able to successfully support mammalian embryo development. Additionally, use of combinatorial buffer systems may offer an advantage over mono-buffered handling media by reducing individual buffer concentrations and toxicity concerns (Swain and Pool 2009).

pH and TemperatureIt is often not appreciated that temperature affects

buffering capacity (pKa) and actual pH of Good’s-buffered media. (Swain and Pool 2009, also Table 3, Figure 4). Thus, one needs to be aware of the working temperature when selecting a specific buffer. One practical consideration is how much the pH measurements change in response to temperature. Both MOPS and HEPES display approximately equal changes in pKa in response to temperature changes between 25°C and 37°C (0.18 and 0.17, respectively) (Sigma). However, other buffers change more significantly. This may be important for procedures with dramatic changes in temperature, such as cryopreservation. Interestingly, in preliminary findings, MOPS was reported to be superior to HEPES when used for vitrification although the exact reason for this remains unclear and the comparison was not made during the same time period (El-Danasouri et al. 2004). Regardless of the buffer chosen, it is crucial to maintain an appropriate and constant temperature to avoid changes in media pH. Furthermore, temperature can also impact pH electrode readings (Electrode Effect). Therefore, correct calibration, either warming the pH electrode/apparatus to the measured temperature, or using a temperature compensation probe is important to ensure accuracy.

INSTRUCTIONS FOR AUTHORS

E-mail articles for The Journal of Clinical

Embryology™ as Word documents, double-spaced,

in any ART journal format, with all authors’

professional affiliations and e-mails. FAX or mail

signed compliance with conventional standards

of authorship and originality.

Please contact us prior to submission for

information regarding formats and requirements.

Keep paragraphs short. Skip 2 spaces

between paragraphs. Articles 1,500-3,000 words

(6-12 pages), shorter for abstracts, lab tips and

letters (signed, return mail and e-mail addresses).

Next article due date: July 7, 2010 for Vol.

13, Issue 3 (Fall 2010).

Email to: [email protected]

Office Phone: (352) 331-5235


21

Figure 4. Changes in temperature (■) result in changes in pH (○) of Good’s buffered media. A) As the temperature rises, the pH of 20mM HEPES buffered media drops. B) When temperature is maintained constant, pH remains stable. The same trend holds true for other buffers, such as MOPS.

Table 3. Common Good’s buffers and their effective buffering ranges. A.) Changes in temperature alter buffering capacity of these pH buffers (data obtained from Sigma-Aldrich) B) pH of handling media changes with temperature.

Measurement of pHeThe importance of pH stability during IVF should

now be readily apparent. Thus, measuring pH properly is critical to ensure cells are being exposed to appropriate conditions. Many labs use Fyrite to measure CO2 levels in their incubator as a means of quality control. From above, we know that the purpose of this exercise is to indirectly measure pHe. However, this approach is inaccurate at best. Fyrite readings often fluctuate and are not overly reliable indicators of pHe. This was eloquently demonstrated by data previously presented in this publication (Pool 2004, Figure 5).

Figure 5. Demonstration of the fluctuation and inaccuracy of fyrite as an indicator of pH. During week 11, fyrite did not accurately identify when pH fell out of range (adapted from Pool 2004).

A more accurate method of measuring pH is to do so directly. This first requires the appropriate pH probe and calibration. Composition of media can affect probe readings and some pH probes work better than others in solutions with organic buffers or protein content (Pooler et al. 1998). It is often recommend that a double-junction glass calomel electrode be used for media with protein or organic buffers. Cleaning or replacement of probes at set intervals helps ensure rapid and accurate readings. Importantly, it is recommended that the validity of readings be substantiated before implementing a new probe into clinical use, especially if


22

one switches probe technology. Some newer technology probes can vary up to 0.2 pH units from other probes in the same solution (Pooler et al. 1998; Quinn and Cooke 2004) .

Calibration entails using two standard solutions that bracket your target pH. In the case of IVF, solutions of pH 7 and 10 are recommended. Both these standards should be warmed to 37°C or one’s pH meter should utilize temperature compensation to ensure accuracy. Once the probe has been calibrated properly, one should confirm accurate pH readings before taking a measurement. This can be accomplished by placing the probe back into pH solution 7 to verify the appropriate pH is recorded or by measuring another standard such as pH7.4.

Ideally, one would measure pH in the incubator being used, under exact conditions to which the cells are exposed (volume, etc). Although specialized pH meters now exist to allow this, some of these newer technology based units are not overly accurate under testing conditions (Pooler et al. 1998), and most are very expensive. Thus, a bench top pH meter is sufficient in measuring test tubes of media removed from the incubator. As long as tubes are capped immediately upon removal and quickly measured, accurate readings can be obtained (Pool 2004). Alternatively, though also expensive, another highly accurate method of determining media pH is through the use of a blood gas analyzer.

It should now be clear that because of the dynamic nature of pHe and the effects of protein additives, lot-to-lot variations in media, as well as other variables; the ability to accurately adjust CO2 concentrations in the incubator offers the greatest ability to provide the most control over pHe. Thus, although the use of pre-mixed gas cylinders, in conjunction with dessicator jars, modular chambers, or small bench-top incubators, may offer advantages in regard to maximizing gas recovery times, they do not offer the same flexibility in fine-tuning pHe in response to slight environmental fluctuations. Therefore, it is important, when employing pre-mixed gases, to be able to verify that the pHe of each medium (containing protein) projected for clinical use be stable within an acceptable pH range before implementation. Furthermore, regular checks are required to verify that pHe remains within this range throughout the use of an individual tank, as media lot #

may change over the course of use.

Future DirectionsThough likely media dependent, there remains a gap

in our knowledge regarding the ideal pHe in which to culture cells during various steps of IVF. There may be differential pHe conditions required for optimal in vitro oocyte maturation , fertilization and various stages of developing embryos. Until properly controlled studies are performed, debate will remain as to the ideal conditions necessary to support these various events What is inarguable is that stabilization of environmental parameters, such as pH, are crucial for optimized culture conditions. Potential for mitigating these damaging oscillations lies in the use of various buffering systems. Importantly, even brief exposure to sub-optimal handling media and culture conditions can compromise resulting embryo quality (Gardner and Lane 1996; Farrell and Bavister 1984; Escriba et al. 2001; Palasz et al. 2008). This remains an area of potential improvement. The benefits obtained by using combinatorial pH buffers within handling media or during culture within the incubator environment, may reside in allowing the selection of a specific pKa with optimal buffering at various temperatures, while lowering individual buffer concentrations. Continued research in this field, along with further examination of various molecular endpoints in order to tease out differences between varying treatments, will aid in these endeavors. Additionally, improved methods of monitoring and adjusting pH are becoming available through the use of real-time pH measurement devices that can be used within the incubator. As this technology becomes more affordable and reliable, one could envision its use in conjunction with newer emerging technology, such as microfluidics, where optical micro or nano-sensors (Clark et al. 1999; Baldini et al. 2007) could detect pH fluctuations and allow for instant adjustment when parameters fall out of range. n

ReferencesAli, J., W. K. Whitten, et al. (1993). “Effect of culture systems on

mouse early embryo development.” Hum Reprod 8(7): 1110-4.Babcock, D. F. and D. R. Pfeiffer (1987). “Independent elevation

of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms.” J Biol Chem 262(31): 15041-7.


23

Babcock, D. F., G. A. Rufo, Jr., et al. (1983). “Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm.” Proc Natl Acad Sci U S A 80(5): 1327-31.

Baldini, F., A. Giannetti, et al. (2007). “Optical sensor for interstitial pH measurements.” J Biomed Opt 12(2): 024024.

Barnett, D. K. and B. D. Bavister (1996). “Inhibitory effect of glucose and phosphate on the second cleavage division of hamster embryos: is it linked to metabolism?” Hum Reprod 11(1): 177-83.

Barnett, D. K., M. K. Clayton, et al. (1997). “Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria.” Mol Reprod Dev 48(2): 227-37.

Barr, K. J., A. Garrill, et al. (1998). “Contributions of Na+/H+ exchanger isoforms to preimplantation development of the mouse.” Mol Reprod Dev 50(2): 146-53.

Bavister, B. (1981). Analysis of culture media for in vitro fertilization and criteria for success. Fertilization and Early Development In Vitro. L. Mastroianni and J. Biggers. New York, Plenum Press: 41-60.

Bavister, B. D. and J. M. Squirrell (2000). “Mitochondrial distribution and function in oocytes and early embryos.” Hum Reprod 15 Suppl 2: 189-98.

Behr, B. R., C. J. Stratton, et al. (1990). “In vitro fertilization (IVF) of mouse ova in HEPES-buffered culture media.” J In Vitro Fert Embryo Transf 7(1): 9-15.

Bhattacharyya, A. and R. Yanagimachi (1988). “Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer.” Gamete Res 19(2): 123-9.

Brinster, R. L. (1965). “Studies on the Development of Mouse Embryos in Vitro. I. The Effect of Osmolarity and Hydrogen Ion Concentration.” J Exp Zool 158: 49-57.

Brook, P. F., J. Lawry, et al. (1996). “Measurement of intracellular pH in human spermatozoa by flow cytometry with the benzo[c]xanthene dye SNAFL-1: a novel, single excitation, dual emission, molecular probe.” Mol Hum Reprod 2(1): 18-25.

Byrd, S. R., G. Flores-Foxworth, et al. (1997). “In vitro maturation of ovine oocytes in a portable incubator.” Theriogenology 47(4): 857-64.

Christen, R., R. W. Schackmann, et al. (1982). “Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocentrotus purpuratus.” J Biol Chem 257(24): 14881-90.

Clark, H. A., R. Kopelman, et al. (1999). “Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.” Anal Chem 71(21): 4837-43.

Cross, N. L. and P. Razy-Faulkner (1997). “Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone.” Biol Reprod 56(5): 1169-74.

Dale, B., Y. Menezo, et al. (1998). “Intracellular pH regulation in the human oocyte.” Hum Reprod 13(4): 964-70.

Downs, S. M. and A. M. Mastropolo (1997). “Culture conditions affect meiotic regulation in cumulus cell-enclosed mouse oocytes.” Mol Reprod Dev 46(4): 551-66.

Edwards, L. J., D. A. Williams, et al. (1998). “Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH.” Hum Reprod 13(12): 3441-8.

Edwards, L. J., D. A. Williams, et al. (1998). “Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids.” Mol Reprod Dev 50(4): 434-42.

El-Danasouri, I., H. Selman, et al. (2004). “Comparison of MOPS and HEPES buffers during vitrification of human embryos.” Hum Reprod 14: i136.

Elrod, C. C. and W. R. Butler (1993). “Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein.” J Anim Sci 71(3): 694-701.

Emmens, C. W. (1947). “The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations.” J Physiol 106(4): 471-81.

Erdogan, S., G. FitzHarris, et al. (2005). “Mechanisms regulating intracellular pH are activated during growth of the mouse oocyte coincident with acquisition of meiotic competence.” Dev Biol 286(1): 352-60.

Escriba, M. J., M. A. Silvestre, et al. (2001). “Comparison of the effect of two different handling media on rabbit zygote developmental ability.” Reprod Nutr Dev 41(2): 181-6.

Farrell, P. S. and B. D. Bavister (1984). “Short-term exposure of two-cell hamster embryos to collection media is detrimental to viability.” Biol Reprod 31(1): 109-14.

Ferguson, W. J., K. I. Braunschweiger, et al. (1980). “Hydrogen ion buffers for biological research.” Anal Biochem 104(2): 300-10.

Fitzharris, G. and J. Baltz (2009). “Regulation of intracellular pH during oocyte growth and maturation in mammals.” Reproduction.

Fitzharris, G. and J. M. Baltz (2006). “Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth.” Development 133(4): 591-9.

FitzHarris, G., V. Siyanov, et al. (2007). “Granulosa cells regulate

NOTE TO ADVERTISERS

Please request 2010 advertising rates for both

hardcopy advertising and website banner ads.

A discount of 10% is available with a prepaid

agreement covering four (4) consecutive issues.

For more information, please contact The Journal

of Clinical Embryology™ advertising office:

[email protected] • (352) 331-5235

— The Publisher


24

oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms.” Development 134(23): 4283-95.

Florman, H. M., R. M. Tombes, et al. (1989). “An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis.” Dev Biol 135(1): 133-46.

Gardner, D. K. and M. Lane (1996). “Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters.” Hum Reprod 11(12): 2703-12.

Gibb, C. A., P. Poronnik, et al. (1997). “Control of cytosolic pH in two-cell mouse embryos: roles of H(+)-lactate cotransport and Na+/H+ exchange.” Am J Physiol 273(2 Pt 1): C404-19.

Good, N. E. and S. Izawa (1972). “Hydrogen ion buffers.” Methods Enzymol 24: 53-68.

Good, N. E., G. D. Winget, et al. (1966). “Hydrogen ion buffers for biological research.” Biochemistry 5(2): 467-77.

Graves, C. N. and J. D. Biggers (1970). “Carbon dioxide fixation by mouse embryos prior to implantation.” Science 167(924): 1506-8.

Hagen, D. R., R. S. Prather, et al. (1991). “Development of one-cell porcine embryos to the blastocyst stage in simple media.” J Anim Sci 69(3): 1147-50.

Hamamah, S. and J. L. Gatti (1998). “Role of the ionic environment and internal pH on sperm activity.” Hum Reprod 13 Suppl 4: 20-30.

Hamamah, S., E. Magnoux, et al. (1996). “Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone.” Mol Hum Reprod 2(4): 219-24.

Hershlag, A. and H. Feng (2001). “The effect of CO2 concentration and pH on the in vitro development of mouse embryos.” Fertility Magazine 4: 21-22.

Herubel, F., S. El Mouatassim, et al. (2002). “Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development.” Zygote 10(2): 175-81.

Hugentobler, S., D. G. Morris, et al. (2004). “In situ oviduct and uterine pH in cattle.” Theriogenology 61(7-8): 1419-27.

Iwasaki, T., E. Kimura, et al. (1999). “Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes.” Theriogenology 51(4): 709-20.

John, D. P. and A. A. Kiessling (1988). “Improved pronuclear mouse embryo development over an extended pH range in Ham’s F-10 medium without protein.” Fertil Steril 49(1): 150-5.

Kane, K. K., K. W. Creighton, et al. (2002). “Effects of varying levels of undegradable intake protein on endocrine and metabolic function of young post-partum beef cows.” Theriogenology 57(9): 2179-91.

Krisher, R. L. and B. D. Bavister (1999). “Enhanced glycolysis after maturation of bovine oocytes in vitro is associated with increased developmental competence.” Mol Reprod Dev 53(1): 19-26.

Lane, M., J. M. Baltz, et al. (1998). “Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter.” Biol Reprod 59(6): 1483-90.

Lane, M., J. M. Baltz, et al. (1999). “Bicarbonate/chloride

Andrology Products

Nidacon PureSperm

Leja Slide — Semen analysis chamber

Vas-Check (R) Post — Vasectomy counting chamber

Bioscreen — Assay kits

QUICK III — Morphology stain

Testsimplets — Prestained slides for cell morphology

Accu-Beads — Quality control method for semen analysis

Hamilton-Thorne — Automated sperm analyzer diAgnostics Pregnancy Test Kits

cryotech Products MVE — Cryo- preservation tanks

IMV — Freezing supplies

consumAbles Pristine Gloves — Powder-free surgical gloves

Oocyte Retrieval Needles

Embryo Transfer Catheters

IUI Catheters

Falcon Labware

Nalgene & Nunc — Plastics & labware

Male-Factor Paks

equiPment Centrifuges

Microscopes

nidAcon AnimAl Art Products Nidacon EquiPure™ Top and Bottom Layer

Nidacon BoviPure™ Top and Bottom Layer

Today’s male infertility clients demand a lot from their IVF specialists. So you need a supplier you can count on for the advanced reproductive technology products you need, when you need them. Whether it’s Halosperm® DNA fragmentation kits, Nidacon PureSperm® isolation media, Leja® Standard Count disposable counting chambers, or any of our complete line of male infertility products and services, Spectrum is your one-stop source. For free samples call 800-358-7509, or email your request to [email protected].

Your clients expect flawless

service. So do ours.

yourPurespermsource.com800• 358 •7509

1532 Chablis Road Suite 101 Healdsburg, CA 95448 707.433.5383 f

T E C H N O L O G I E S

The products you need . . . the service you expect.

tm


25

exchange regulates intracellular pH of embryos but not oocytes of the hamster.” Biol Reprod 61(2): 452-7.

Lane, M., J. M. Baltz, et al. (1999). “Na+/H+ antiporter activity in hamster embryos is activated during fertilization.” Dev Biol 208(1): 244-52.

Lane, M. and B. D. Bavister (1999). “Regulation of intracellular pH in bovine oocytes and cleavage stage embryos.” Mol Reprod Dev 54(4): 396-401.

Lane, M. and D. K. Gardner (2000). “Regulation of ionic homeostasis by mammalian embryos.” Semin Reprod Med 18(2): 195-204.

Lane, M., T. E. Ludwig, et al. (1999). “Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.” Mol Reprod Dev 54(4): 410-7.

Lane, M., E. A. Lyons, et al. (2000). “Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH.” Hum Reprod 15(2): 389-94.

Leclerc, C., D. Becker, et al. (1994). “Low intracellular pH is involved in the early embryonic death of DDK mouse eggs fertilized by alien sperm.” Dev Dyn 200(3): 257-67.

Lee, M. A. and B. T. Storey (1986). “Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction.” Biol Reprod 34(2): 349-56.

Lenart, P., C. P. Bacher, et al. (2005). “A contractile nuclear actin network drives chromosome congression in oocytes.” Nature 436(7052): 812-8.

Lepe-Zuniga, J. L., J. S. Zigler, Jr., et al. (1987). “Toxicity of light-exposed Hepes media.” J Immunol Methods 103(1): 145.

Mahadevan, M. M., J. Fleetham, et al. (1986). “Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, hepes, or phosphate.” J In Vitro Fert Embryo Transf 3(5): 304-8.

Marquez, B. and S. S. Suarez (2007). “Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx.” Biol Reprod 76(4): 660-5.

Morgia, F., M. Torti, et al. (2006). “Use of a medium buffered with N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertilization.” Fertil Steril 85(5): 1415-9.

Nagai, S., T. Mabuchi, et al. (2006). “Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence.” Tohoku J Exp Med 210(2): 137-44.

Navarro, B., Y. Kirichok, et al. (2007). “KSper, a pH-sensitive K+ current that controls sperm membrane potential.” Proc Natl Acad Sci U S A 104(18): 7688-92.

Ozawa, M., T. Nagai, et al. (2006). “Successful pig embryonic development in vitro outside a CO2 gas-regulated incubator: effects of pH and osmolality.” Theriogenology 65(4): 860-9.

Palasz, A. T., P. B. Brena, et al. (2008). “The effect of different zwitterionic buffers and PBS used for out-of-incubator procedures during standard in vitro embryo production on development, morphology and gene expression of bovine embryos.” Theriogenology 70(9): 1461-70.

Phillips, K. P. and J. M. Baltz (1999). “Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development.” Dev Biol 208(2): 392-405.

Phillips, K. P., M. C. Leveille, et al. (2000). “Intracellular pH

regulation in human preimplantation embryos.” Hum Reprod 15(4): 896-904.

Phillips, K. P., M. A. Petrunewich, et al. (2002). “The intracellular pH-regulatory HCO3-/Cl- exchanger in the mouse oocyte is inactivated during first meiotic metaphase and reactivated after egg activation via the MAP kinase pathway.” Mol Biol Cell 13(11): 3800-10.

Pholpramool, C. and G. Chaturapanich (1979). “Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa.” J Reprod Fertil 57(1): 245-51.

Pool, T. (2004). “Optimizing pH in Clinical Embryology.” The Clinical Embryologist 7(3): 1-17.

Pooler, P. M., M. L. Wahl, et al. (1998). “pH measurement: a comparison of results using glass membrane electrodes vs solid-state electrodes as a function of solution composition.” Anal Biochem 256(2): 238-40.

Quinn, P. and S. Cooke (2004). “Equivalency of culture media for human in vitro fertilization formulated to have the same pH under an atmosphere containing 5% or 6% carbon dioxide.” Fertil Steril 81(6): 1502-6.

Quinn, P. and R. G. Wales (1971). “Fixation of carbon dioxide by pre-implantation mouse embryos in vitro and the activities of enzymes involved in the process.” Aust J Biol Sci 24(6): 1277-90.

Quinn, P. and R. G. Wales (1974). “Fixation of carbon dioxide by preimplantation rabbit embryos in vitro.” J Reprod Fertil 36(1): 29-39.

Regula, C. S., J. R. Pfeiffer, et al. (1981). “Microtubule assembly and disassembly at alkaline pH.” J Cell Biol 89(1): 45-53.

Schmidt, J., C. Mangold, et al. (1996). “Membrane responses evoked by organic buffers in identified leech neurones.” J Exp Biol 199(Pt 2): 327-35.

Spindler, R. E., B. S. Pukazhenthi, et al. (2000). “Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro.” Mol Reprod Dev 56(2): 163-71.

Squirrell, J. M., M. Lane, et al. (2001). “Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos.” Biol Reprod 64(6): 1845-54.

Steel, T. and J. Conaghan (2008). “pH equilibration dynamics of culture medium under oil.” Fertil Steril 89(suppl 2): s27.

Stellwagen, N. C., A. Bossi, et al. (2000). “DNA and buffers: are there any noninteracting, neutral pH buffers?” Anal Biochem 287(1): 167-75.

Summers, M. C. and J. D. Biggers (2003). “Chemically defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues.” Hum Reprod Update 9(6): 557-82.

Swain, J. and T. Pool (2009). “Supplementation of sequential embryo culture medium with synthetic organic buffers supports development of mouse embryos in an elevated CO2 environment.” RBM Online 16(suppl 4).

Swain, J. E. and T. B. Pool (2009). “New pH-buffering system for media utilized during gamete and embryo manipulations for assisted reproduction.” Reprod Biomed Online 18(6): 799-810.

Wersinger, C., G. Rebel, et al. (2001). “Characterisation of taurine uptake in human KB MDR and non-MDR tumour cell lines

(continued on page 28)

Vitrol i fe Inc. , 3601 South Inca Street, Englewood, Colorado 80110, USA, Tel : +1-303-762-1933, Fax: +1-303-762-7084, E-mail : fert i l i ty .us@vitrol i fe.com, www.vitrol i fe.com

Life just got easier.

Introducing Rapid-i™ Vitrifi cation System. The world’s fi rst integrated closed

vitrifi cation system.

We sat back and looked at the entire vitri-fi cation process. From A to Z. We wanted to create a system that provided maximum safety for embryos, while making your job as easy as possible. And here it is.

Tools and media are designed to work seamlessly together – in what we call the SmartBox. It’s even got a magnet inside to keep the straws in place. Believe us, we tried to think of everything.

© V

itro

life

1.10

0426

.3

JCE Rapid may10 spread.indd 2 2010-04-27 17.33.29


28

Ashok Agarwal, Ph.D. HCLDStaff, Glickman Urological Institute, and Departments of Obstetrics/Gynecology, Anatomic Pathology, and Immunology Case Western Reserve UniversityCleveland Clinic Foundation Cleveland, Ohio 44195 [email protected]

David E. Battaglia, Ph.D., HCLD/ELDOHSU Fertility ConsultantsOregon Health and Science UniversityPortland, [email protected]

Barry D. Bavister, Ph.D.Adjunct ProfessorUniversity of Puerto RicoMedical Sciences Campus San Juan, Puerto Rico Adjunct ProfessorDept. of Obstetrics & Gynecology Wayne State University, Detroit, MI, USA [email protected]

Barry Behr, PhD, HCLD Director, IVF/ART Laboratory Co-Director, Stanford Fertility and Reproductive Medicine Centers REI/IVF Program Associate Professor, Department of OB/[email protected]

Carol Brenner, PhDDepartments of Obstetrics & Gynecology and PhysiologyWayne State UniversitySchool of MedicineDetroit MI [email protected]

Grace Centola, Ph.D., H.C.L.D.Professional ReproLab ConsultingMacedon, New [email protected]

Kathryn J. Go, Ph.D. HCLDReproductive Science CenterOne Forbes RoadLexington, MA [email protected]

David L. Hill, Ph.D. HCLDART Reproductive CenterBeverly Hills, [email protected]

David Mortimer, Ph.D.President, Oozoa BiomedicalBox 93012 Caulfeild Village RPO, West Vancouver, BC, V7W [email protected]

Thomas B. Pool, Ph.D. HCLDFertility Center of San AntonioSan Antonio, [email protected]

Richard G. Rawlins, Ph.D. HCLDRush Centers for AdvancedReproductive CareDept OB/GYNRush Medical CenterChicago, IL [email protected]

Alan Thornhill, Ph.D. HCLD Scientific Director The London Bridge Fertility, Gynaecology and Genetics Centre One St Thomas Street, London Bridge, London SE1 [email protected]

Kenneth C. Drury, Ph.D. HCLDDept OB/GYNUniversity of Florida College of MedicineGainesville, FL [email protected]

Fred M.W. ZanderPublisher

The Journal Of Clinical Embryology™ Editorial Board

in culture.” Anticancer Res 21(5): 3397-406.Zander-Fox, D., M. Mitchell, et al. (2008). “Repercussions of a

transient decrease in pH on embryo viability and subsequent fetal development. .” Reprod Fertil Dev 20.

Zeng, Y., J. A. Oberdorf, et al. (1996). “pH regulation in mouse sperm: identification of Na(+)-, Cl(-)-, and HCO3(-)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation.” Dev Biol 173(2): 510-20.

Zhao, Y. and J. M. Baltz (1996). “Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo

development.” Am J Physiol 271(5 Pt 1): C1512-20.Zhao, Y., P. J. Chauvet, et al. (1995). “Expression and function

of bicarbonate/chloride exchangers in the preimplantation mouse embryo.” J Biol Chem 270(41): 24428-34.

Zhu, Z. Y., D. Y. Chen, et al. (2003). “Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes.” Biol Reprod 68(3): 943-6.

Zigler, J. S., Jr., J. L. Lepe-Zuniga, et al. (1985). “Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium.” In Vitro Cell Dev Biol 21(5): 282-7.

(continued from page 25)


29

Techniques to achieve low oxygen tension for culture of human embryos

Presented at: 11th Annual Embryologist’s Summit, Madison, Wisconsin, May 1, 2010

Michael L. Reed, Ph.D. HCLD (ABB)Center for Reproductive Medicine of New Mexico

Albuquerque, New [email protected]

The debate over whether human embryos should be maintained in vitro under physiological oxygen concentrations, similar

to that estimated for in vivo oviduct and uterine tissue (e.g. approximately 5 to 10%), or whether they should be cultured using an atmospheric oxygen tension of around 20%, is ongoing and largely unresolved. The premise of this presentation is that the embryologist desires to culture human embryos at physiologic oxygen concentrations, and would like to explore the mechanical options available to achieve this endpoint.

Discussion on the effects of oxygen tension on embryonic development in vitro must include consideration for the processes of oxidative stress, defined here as the balance (or conflict) between pro-oxidant stressors (e.g. reactive oxygen species) and anti-oxidant capacity. In vivo, the essential anti-oxidative capacity includes that inherent in the oocyte/embryo and of the oviduct and uterine environment, where in vitro, the latter processes are absent, replaced to some degree by the appropriate use of anti-oxidants in culture media and/or minimization of pro-oxidative stressors, where care is taken to optimize oxygen tension, minimize exposure to visible light, minimize fluctuations in temperature and humidity, and so on. The effect(s) of in vitro oxidative processes on reproduction are thought to include damage to sperm, oocyte, and embryo lipid membranes, damage to nuclear and mitochondrial DNA, altered gene and epigenetic expression, apoptosis, and reduced embryonic competence (for review, see 1-5).

A literature review on oxygen tension and embryo culture reveals a large number of publications stemming

from the animal sciences, and respectively fewer publications from human clinical medicine. Animal studies can be, by their very nature, more complex, involving more robust study designs with relatively homogeneous study populations, while human in vitro embryo research is limited primarily to evaluation of observational data, such as numbers of cleavage divisions, embryo morphology, blastocyst formation, and clinical outcomes, in a very heterogeneous population. The bulk of the non-human animal data points towards benefits of embryo culture with lower, physiological oxygen concentrations, while the data with human clinical IVF has been less definitive.

For example, a recent, well-designed study (6) found that physiological oxygen culture improved clinical outcomes, while another recent, well-designed study (7) found no differences in clinical outcomes, comparing culture under physiological oxygen to atmospheric oxygen. The main difference between these two studies was that in the latter study, embryos were cultured out to day three in atmospheric oxygen and then assigned embryos to either atmospheric or physiological oxygen concentrations for growth to day five, while in the former study, embryos were cultured in the assigned oxygen atmosphere from fertilization onward. These studies are presented as examples, regarding the effects of oxygen tension on human IVF outcome, where the ‘truth’ is more likely to be confounded by study design, e.g. transfer on day 2 or 3 versus day five or six, varied culture techniques (including or excluding co-culture), culture media, incubation systems, and variables inherent to the population being studied.

What is interesting, however, is that regardless of the source material, e.g. non-human or human embryos,


30

there do not appear to be any studies that claim in vitro culture of embryos at physiological oxygen tension is detrimental to development or other gestational measures.

In the event that the IVF clinic desires to adopt the methods for, and/or the mechanical infrastructure for culture of embryos at reduced oxygen tension, the decision process can begin by asking two fundamental questions:

What are the base operating costs for instituting and maintaining an embryo culture program that utilizes reduced oxygen tension?

A cost analysis is recommended, which, for larger programs, will likely demonstrate that use of nitrogen gas cylinders for triple-mix incubators will be cost prohibitive, leaving nitrogen gas generators and vented nitrogen gas from liquid nitrogen dewars competing as the more cost-effective methods. The costs for implementing low oxygen culture will also depend on whether or not the clinic is trying to adapt using incubators already in place, or replacing existing incubators with newer units that can process multiple gasses, or building a new facility that takes into account not only new incubators but includes construction to accommodate a gas generator. One example of a simple cost comparison is presented in Table 1. Active negotiations with local gas distributors can help to reduce costs, as there are additional fees to consider, e.g. delivery fees, tank rentals, hazard transportation fees, gas certification and administrative fees, to name a few.

Will culture of human embryos at reduced oxygen tension enhance embryo development and epigenetic normalcy, and improve the ability of an embryo to implant and proceed to term?

Any changes to existing protocols and procedures should include an evaluation of the current clinical and laboratory goals, in terms of embryology metrics and patient outcomes. Is the desired endpoint an increase in clinical pregnancies, improved embryo quality, an increase in the number of embryos developing to blastocyst stage for transfer or cryopreservation, or decreased spontaneous pregnancy loss, for example? Depending on the nature and size of the program, some of these, and other variables, may not be easy to analyze in the short term, requiring a level of commitment to

the process, and faith in the science behind the changes. Within patient, sibling embryo comparisons are helpful, minimizing the impact of between-patient variation.

Aside from the monetary cost of pursuing embryo culture at physiological oxygen concentrations, there are questions pertinent to the mechanics of the culture environment. What is the oxygen concentration at or near to the embryo in vitro, under 5% oxygen? Is 5% the ideal oxygen concentration for the embryos in vitro? Static culture techniques, e.g. microdrop or test tube culture, where there are no mechanical devices used to rock or otherwise move the dishes during culture or are not related to the microfluidic culture techniques, have little opportunity for active movement of solutes to or away from embryos, and in fact, oxygen, potassium, and calcium gradients have been detected out to beyond 50 micrometers from mouse embryos, by micro-probe analysis (8,9). Also, mathematical modeling of oxygen diffusion under static culture conditions suggests that diffusion alone is adequate to provide oxygen to embryos in vitro, although the oxygen tension directly next to the embryo (depending upon the stage of development) may be slightly hypoxic (10,11). Two studies, using rabbit (12) and cattle embryos (13) cultured under oxygen concentrations ranging from 1.0% to 20.0% and 2.5% to 20.0%, respectively, demonstrated optimal development at 5% O2, but interestingly, rabbit embryos were more tolerant of very low oxygen concentrations, e.g. 1% oxygen, while cattle embryos were less tolerant of 2.5% oxygen. Another study (14), evaluating sheep and cattle embryo development found that 8% oxygen was optimal. The take home message remains, that reducing oxygen tension from atmospheric to physiological concentrations can be beneficial.

There have been no similar studies using human embryos, where a range of oxygen concentrations have been examined, however it is clear that embryonic competency is demonstrated at both 5% oxygen and atmospheric (≈20%) oxygen concentrations. And there are anecdotal communications to the author stating that embryo culture can be carried out very successfully at 10% oxygen, reducing pro-oxidant exposure while reducing nitrogen gas consumption (and the associated costs) by the incubators.

Cell culture incubators are manufactured in two basic formats: single and multiple gas units. Single gas


31

incubators are traditionally used with atmospheric air, the single gas being carbon dioxide, used to modulate culture medium pH. Oxygen concentrations in this incubator will depend on the regional atmospheric oxygen tension, around 20% across the continental US. The small, bench-top incubators are designed for use with a single gas; they can be used with CO2 and ambient air, or they can be used with a source of mixed gasses, for example, from a certified gas cylinder. Multiple gas cell culture incubators are designed for use, traditionally, with nitrogen and carbon dioxide source gasses, where the oxygen concentration is controlled by the input of nitrogen gas to physically displace the oxygen. Multiple gas incubators are inefficient in terms of nitrogen use, in that they consume large volumes of nitrogen, and as such they can be more expensive to operate. Nitrogen gas can be sourced from certified gas cylinders (the more expensive option), or from gas vented off of a large, high pressure liquid nitrogen tank, or derived from atmospheric air via a nitrogen generator. A more cumbersome, but less costly alternative is to us glass or plastic jars filled under low pressure with a certified source of mixed gasses, typically from a single cylinder, custom mixed to meet the requirements of the culture system, e.g. 5%O2, 5%CO2, 90%N2. The gassed, and temperature equilibrated, individual culture chambers are then placed within a standard cell culture incubator. The

limitation to using certified gas mixes is that you cannot change the mix without having another cylinder prepared, while traditional ‘triple-mix’ gas incubators allow for fairly rapid changes to the gas mix by the embryologist.

An additional list of references, though not comprehensive, has been provided in Appendix 1 as a resource for further reading on this discussion, and related topics. n

References1. Guérin P, El Mouatassim S, Ménézo Y. Oxidative stress and

protection against reactive oxygen species in the pre-implantation embryo and its surroundings. 2001 Hum Reprod Update 7:175-189

2. Bedaiwy MA, Falcone T, Mohamed MS, Aleem AA, Sharma RK, Worley SE, Thornton J, Agarwal A. Differential growth of human embryos in vitro: role of reactive oxygen species. 2004 Fertil Steril 82:593-600

3. Harvey AJ. The role of oxygen in ruminant preimplantation embryo development and metabolism. 2007 Animal Reprod Sci 98:113-128

4. du Plessis, SS, Makker, K, Desai, NR, Agarwal, A. The impact of oxidative stress on in vitro fertilization. 2008 Expert Rev. Obstet. Gynecol 3:539-554

5. Krajcir N, Chowdry H, Gupta S, Agarwal A. Female Infertility and Assisted Reproduction: Impact of Oxidative Stress. Current Woman’s Health Review. 2008; 4: 9-15

6. Meintjes M, Chantilis SJ, Douglas JD, Rodriguez AJ, Guerami AR, Bookout DM, Barnett BD, Madden JD. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. 2009 Hum

Table 1. Estimated gas costs1 for operating 7 standard cell culture incubators non-stop for one year at Center for Reproductive Medicine of New Mexico

Incubator Source of gas Culture environment Base cost estimate/yr Notes

7 single gas incubators CO2 cylinders CO2 only $600.00 Per year

CO2 cylinders6/5/89 cylinders

CO2 & triple mix cylinder, with jars

$600.00$900.00

Per year – current CRMNM configuration

7 ‘triple mix’ incubators

LN2 dewar off-gasCO2 cylinders Triple mix $9,500.00

$600.00 Per year

N2 gas generator*CO2 cylinders Triple mix ↓each year

$600.00One time cost* CO2 per year

N2 cylindersCO2 cylinders Triple mix $30,000.00

$600.00 Per year

1Assumptions based on the authors’ local costs, without bulk contract negotiations, and a non-binding phone quote for a nitrogen gas generator; does not include delivery fees, DOT fees, miscellaneous attachments, regulators, and so on. These figures do not take into account purchase of new incubators.


32

Reprod 24:300-3077. Nanassy L, Peterson CA, Wilcox AL, Peterson CM, Hammoud

A, Carrell DT. Comparison of 5% and ambient oxygen during days 3-5 of in vitro culture of human embryos. 2010 Fertil Steril 93:579-585

8. Trimarchi JR, Liu L, Porterfield DM, Smith PJ, Keefe DL. A non-invasive method for measuring preimplantation embryo physiology. 2000 Zygote 8:15-24

9. Trimarchi JR, Liu L, Smith PJ, Keefe DL. Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos. 2000 Biol Reprod 62:1866-1874

10. Byatt-Smith JG, Leese HJ, Gosden RG. An investigation by mathematical modeling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion. 1991 Hum Reprod 6:52-57

11. Baltz JM, Biggers JD. Oxygen transport to embryos in microdrop cultures. 1991 Mol Reprod Dev 28:351-355

12. Li J, Foote RH. Culture of rabbit zygotes into blastocysts in protein-free medium with one to twenty per cent oxygen. 1993 J Reprod Fertil 98:163-7

13. Takahashi Y, Hishinuma M, Matsui M, Tanaka H, Kanagawa H. Development of in vitro matured/fertilized bovine embryos in a chemically defined medium: influence of oxygen concentration in the gas atmosphere. 1996 J Vet Med Sci 58:897-902

14. Thompson JG, Simpson AC, Pugh PA, Donnelly PE, Trevit HR. Effect of oxygen concentration on in-vitro development of preimplantation sheep and cattle embryos. J Reprod Fertil 1990 89:573-578

Appendix 1: Additional readingOxidative stress and reproduction:Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen

species in the pathophysiology of human reproduction. Review article. 2003 Fertil Steril 79:829-843

Agarwal A, Gupta S, Sharma R. Oxidative stress and its implications in female infertility - a clinician’s perspective. 2005 Reprod Biomed Online 11:641-650

Human embryo culture and reduced oxygen:Dumoulin JC, Vanvuchelen RC, Land JA, Pieters MH, Geraedts

JP, Evers JL. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse. 1995 Fert Steril 63:115-119

Dumoulin JC, Meijers CJ, Bras M, Coonen E, Geraedts JP, Evers JL. Effect of oxygen concentration on human in-vitro fertilization and embryo culture. 1999 Hum Reprod 14:464-469

Dumoulin JC, Coonen E, Bras M, van Wissen LC, Ignoul-Vanvuchelen R, Bergers-Jansen JM, Derhaag JG, Geraedts JP, Evers JL. Comparison of in-vitro development of embryos originating from either conventional in-vitro fertilization or intracytoplasmic sperm injection. 2000 Hum Reprod 15:402-409

Catt JW, Henman M. Toxic effects of oxygen on human embryo development. 2000 Hum Reprod 15(suppl 2):199-206

Bahçeci M, Ciray HN, Karagenc L, Uluğ U, Bener F. Effect of oxygen concentration during the incubation of embryos of women undergoing ICSI and embryo transfer: a prospective randomized study. 2005 Reprod Biomed Online 11:438-443

Petersen A, Mikkelsen AL, Lindenberg S. The impact of oxygen tension on developmental competence of post-thaw human embryos.

2005 Acta Obstet Gynecol Scand 84:1181-1184Kea B, Gebhardt J, Watt J, Westphal LM, Lathi RB, Milki AA, Behr

B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization. 2007 Fertil Steril 87:213-216

Anderson AR, Graff KJ, Crain JL. Low oxygen tension and euploidy after preimplantation genetic screening. 2007 Fertil Steril 88(suppl 1):S91

Janzen JMC, Graff D, Anderson S, Matt DW. Comparison of atmospheric oxygen versus low oxygen on human sibling embryo development. 2008 Fertil Steril 90(suppl):S431-S432

Kovacic B, and Vlaisavljevic, V. Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes. 2008 Reprod Biomed Online 17:229-236

Waldenström U, Engström AB, Hellberg D, Nilsson S. Low-oxygen compared with high-oxygen atmosphere in blastocyst culture, a prospective randomized study. 2009 Fertil Steril 91:2461-2465

Ciray HN, Aksoy T, Yaramanci K, Karayaka I, Bahceci M In vitro culture under physiologic oxygen concentration improves blastocyst yield and quality: a prospective randomized survey on sibling oocytes. 2009 Fertil Steril 91(4 Suppl):1459-61

Higdon NL, Herlong MD, Johnson JE, Boone WR. In vitro culture of human embryos: effect of oxygen tension on resulting offspring. 2009 J Clinical Embryology (Fall) 12:6-11

Non-human animal embryo culture and low oxygen: Hallden K, Li J, Carney EW, Foote RH. Increasing carbon

dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes. 1992 Mol Reprod Dev. 33:276-80

Watson AJ, Watson PH, Warnes D, Walker SK, Armstrong DT, Seamark RF. Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere. 1994 Biol Reprod 50:715-724

Fujitani Y, Kasai K, Ohtani S, Nishimura K, Yamada M, Utsumi K. Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos. 1997 J Anim Sci 75:483-489

Farrell PB, Foote RH. Beneficial effects of culturing rabbit zygotes to blastocysts in 5% oxygen and 10% carbon dioxide. 1995 J Reprod Fertil 103:127-30

Lim JM, Reggio BC, Godke RA, Hansel W. Development of in-vitro-derived bovine embryos cultured in 5% CO2 in air or in 5% O2, 5% CO2 and 90% N2. 1999 Hum Reprod 14:458-464

Iwata H, Minami N, Imai H. Postnatal weight of calves derived from in vitro matured and in vitro fertilized embryos developed under various oxygen concentrations. 2000 Reprod Fertil Dev 12:391-396

Choi YH, Love CC, Varner DD, Love LB, Hinrichs K. Effects of gas conditions, time of medium change, and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection. 2003 Theriogenology 59:1219-1229

Karja NWK, Wongsrikeao P, Murakami M, Agung B, Fahrudin M, Nagai T, Otoi T. Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos. 2004 Theriogenology 59:1585-1595

Im GS, Lai L, Liu Z, Hao Y, Wax D, Bonk A, Prather RS. In



33

MicroSecure Vitrification (µS-VTF) Procedure: Optimum simplicity, security, cost-savings and effectiveness

combining FDA-approved products

Expanded from a presentation for the 11th Clinical Embryologist’s Summit Conference(Improving IVF Outcomes from the Laboratory)

Madison WI, May 1, 2010

Mitchel C. Schiewe, PhDSouthern California Institute for Reproductive Sciences (SCIRS), Newport Beach, CA

[email protected]

A pioneering scientific breakthrough in embryo cryopreservation called “vitrification” (VTF) occurred in a series of experiments conducted

in a cold room at the American Red Cross in Bethesda, MD in the mid-1980’s (Fahy et al., 1984; Rall & Fahy, 1985; Fahy, 1986). Using highly concentrated solutes and cryoprotective agents, a “metastable glass phase” could be achieved without ice crystal formation (i.e., vitrification) under rapid cooling conditions (>100°C/min) in LN2. The third generation of vitrification solutions (VS3a), a 6.5M glycerol-based VS, developed by William Rall was effectively applied to mouse and sheep blastocysts with subsequent live births, under room temperature exposure conditions and embryo storage in sealed 0.25 ml straws and plunged into LN2 vapor within 90 sec (Rall et al., 1987, Schiewe, 1989; Schiewe et al., 1991). Throughout the 1990s’ and into the 21st century, several vitrification solutions have evolved and various, novel devices continue to be developed (see review: Vajta and Nagy, 2006) creating a potential quality control (QC) nightmare for the IVF/ART industry in years ahead.

Considering the good VTF results reported by others this decade (Kuwayama, 2007; Liebermann & Tucker, 2004; Yoon et al., 2003), studies have been subject to “technical signature”, i.e., the technical skill of the individual(s) performing the VTF procedures. Unfortunately, the experience of individual programs having poor VTF results and patient outcomes are not reported to magnify the importance of the latter issue. Yet, in informal discussions, we know that technical variation exists between individuals, especially in the early application phase. VTF methods are critically

time-dependent and can involve challenging handling methodologies (e.g., cryo-loops/tops/tips/locs/leafs, EM grids, nylon mesh). One solution to making VTF safer (i.e., less toxic) and simpler has been reported by eliminating DMSO from the standard VTF solution and using 0.25 ml straws, resulting in excellent BL cryo/thaw survival (>90%) and pregnancy success (>55%) across multiple IVF Centers (S3 VTF: Stachecki et al., 2008).

There is growing concern over the long-term cryostorage of samples which are unsealed (i.e., leaky, open container and protected device systems; e.g., cryovials/-loops, cryo-tops/leafs/locs, etc) or improrperly sealed due to potential cross-contamination in LN2 storage tanks (Rall, 2003). Although LN2 vapor phase storage tanks offset these concerns, they are not common to, nor practical, in most clinical IVF Lab settings. The popular and effective, but open-ended Cryo-top method (Kuwayama, 2007; Kuwayama et al., 2005a), was modified into a closed micropipette system marketed by Irvine Scientific as the FDA approved Cryo-tip™ device (Kuwayama et al., 2005b). The Cryo-tip™ does produce a closed, double sealed container, but it has proven to be technically challenging to use (i.e., “technical signature” concept applies) due to aspiration, bubbling and sealing issues, and it lacks complete biosecurity. Alternatively, Isachenko et al. (2005, 2007) developed an aseptic double container system which has proven effective for human oocyte and blastocyst VTF, despite the slower cooling rates (approx. 600 C°/min) caused by an insulation effect. The latter “cut-straw” system (CSS; Isachenko et al., 2007), like the CBS™-HSV “scoop device” (Vanderzwalmen et al, 2003) are still subject to “technical signature” and suboptimal recovery


34

rates (<100%). Furthermore, there are QC/viability concerns over the handling, storage and shipment of vitrified samples (Reed, 2007), which should be taken into account by each laboratory.

MicroSecure VTF (µS-VTF; Schiewe and Anderson, 2008; Schiewe and Fahy, 2008; Schiewe et al., 2009a) has been developed with logic and validation, without commercial influence and marketing pressure. It offers easy, practical handling methods (i.e. high inter-technician repeatability), tamper-proof labeling in a secure aseptic container and an in-situ vapor VTF technique for safe long-term liquid phase LN2 storage. The µS-VTF device uses both the CBSTM embryo straw and sterile denuding tips which already meet FDA HCT/P requirements for Current Good Tissue Practices (CGTPs). The purpose of this paper is to introduce lab personnel to a logical, user-friendly, low cost vitrification system that eliminates QC and regulatory concerns, while sustaining optimum post-thaw results.

Material and MethodsVitrification ProceduresThe successful application of all vitrification

methods, independent of the device used, is dependent on three principle components: clarity of mind (i.e., organization), concentration (i.e., focus on task at hand) and consistency (meticulous, technical repeatability). All paperwork / cryo-records and the preparation of cryo-canes/goblets/straws should be completed prior to initiating VTF. Complete organization {CLARITY} is imperative to avoid any variation in strictly timed dilution and loading/plunging steps, as described below.

mS-VTF DeviceThe microSecure (mS) technique combines the use of

300mm ID flexible, sterile denuding pipette (Flexipet®, COOK®) to load VTF solutions with oocytes or embryos before placing the “VTF tip” into a 0.3ml CBSTM embryo straw for secure, long-term aseptic storage in LN2. The set up involves modifying individual, sterile VTF tips by cutting approximately 2.0 cm from the base of the denuding pipette before securely placing them on Stripper pipetters. Arrange pipetters in an orderly manner. Next, individual colored labels (1.3” x 0.5”; CL-23, GA International Inc) are created, each identifying the Patient Name (last, first), unique ID# (cycle #, e.g., A250-08), straw #, embryo/oocyte ID (# x stage) and Date (mo-d-yr; see Figure 1). The completed label is

attached to a color coded ID rod and inserted into the internal labeling end of the CBSTM straw, and then sealed using a Syms sealer (*important device*: eliminates technical variation). With the labeled end of the CBSTM

straw protruding from its sterile packaging, the nozzle tip is dislodged from the opposite end before use.

Figure 1. Dual color coding of the µS-VTF labels facilitates rapid ID of vitrified samples.

Cryoprotectant In the early testing phases of the µS-VTF system (Study 1 and 2), the Irvine Scientific VTF Cryo and Thaw kits and procedures were used. As we approached clinical application, the laboratory transitioned to using DMSO-free S3 vitrification solutions (Stachecki et al, 2008) in Study 3 and subsequent IRB-approved clinical investigations Due to proprietary rights, the exact concentration of glycol based cryoprotectant, albumin and other macromolecules in the S3 solution are unknown.

Cryodilution and VitrificationSet-up and dilutions are performed at room

temperature (21-24° C). A separate dish (100 x 35mm) is prepared for each patient and for the use of up to 5 mS-devices/ VTF run (approximately 10-15 min). Example, if a donor/patient has 30 mature oocytes to vitrify in groups of 5 oocytes/device, then 2 dishes would be set-up (see Figure 2A). At the top of each dish, a series of 25µl droplets of Hepes-buffered Global®

medium + protein (H-LG+) is used to temporarily hold individual groups of oocytes (n=1-5) or embryos (n=1-2) before beginning VTF dilutions. The larger first drop (50-100ml) to the right side is used as an initial holding drop, changing from the use of an oil-exposed pipette (i.e., right side deposit) to the clean VTF tip(s) (i.e., left side aspiration). The time of dilution in each VTF step may vary based on developmental stage (see Table 1) and the type of VTF solution used, with approximately 120-150ml of VTF solution used in each row (Figure 2A example: 50 ml, 2 x 25 ml, 3 x 10 ml). When transferring oocytes/embryos between droplets, especially between solutions, the pipette is first preloaded with the next


35

solution, then ½ the volume is expelled on the oocyte/embryo before picking it up to move to the next solution. Once in the next droplet, the new solution is aspirated into the tip (i.e., in situ dilution) before expelling the oocyte/embryo. The oocyte/embryo(s) are quickly rinsed in 3 separate areas of the droplet (e.g., a consistent, counter-clockwise triangle pattern is recommended; Figure 2B) before repeating the process in the next droplet. When the last droplet of each solution is reached (Ex: V1), the tip is immediately rinsed and filled with the next solution (Ex: V2) and the Stripper pipetter is returned to a Styrofoam holding rack until it is time to use it again. {CoNsIsTENCY}

In between holding intervals, you can initiate the dilution of another group. With experience, it is possible to dilute up to 5 BL groups, 60 sec apart, within the 5 min V1 and V2 dilution time specified for S3 –BL vitrification solutions. Organization is imperative: consistency in the above mentioned washing / pipetting steps, and placement of stipper pipetters in a rack (e.g., placement on each corner allows you to rotate the rack in a circular process to use the correct tip next).

The above mentioned 60 sec handling time is possible at the end (i.e., LN2 plunge step), due to the simplicity and efficacy of the mS-device loading and sealing steps (below) before direct placement in LN2. When the last straw is plunged, they can be individually placed into a pre-labeled goblet and cane for that patient, before moving on to the next VTF group or patient.

{CoNCENTRATIoN}Upon pipetting the oocyte/embryo(s) into the last

drop of V3, a small volume of the solution is expelled and the oocyte/embryo(s) are loaded into the lower 1/3 of the pipette tip, fully releasing the pipetter plunger. After removing the flexible VTF tip and grasping its base-end, it is wiped onto sterile gauze, using a rolling motion to remove any residual fluid on the outer surface. Simultaneously, the pre-labeled CBSTM straw is grasped with the opposite hand. Next, the clean, oocyte/embryo containing VTF tip is introduced into the CBSTM straw (loading-end first), initially at an angle to facilitate easy entry (Figure 3A). When the tip of the flexible pipette (i.e., VTF tip) enters into the open end of the CBSTM straw, the VTF tip is pushed in a horizontal position. Once inserted, the CBSTM straw was tilted, label end downward, bringing the VTF tip to the inner plug before sealing (Figure 3B). Container sealing is completed by inserting the opened-end of the straw (containing the VTF tip and an air space buffer) into the Syms Sealer, the straw is secured near the sealer to reduce vibration during activation (i.e., sealing). Approximately 5 sec later, the straw was removed and tilted with the newly sealed end downward, the side wall is tapped lightly, if needed, to insure the VTF tip base falls down to the sealed end prior to directly plunging the straw into a dewar flask filled with LN2.

The QC issue involved in drying the VTF tip before placement into the CBSTM straw is a critical step to prevent it from sticking to the inside of the straw during cooling and warming, which would inhibit its rapid release from the opened CBSTM straw post-thaw.

Figure 2. Schematic of Dish set-up and oocyte/embryo pipetting/washing procedure


36

TABLE 1. Dilution Intervals by Developmental Stage, Using S3 Vitrification SolutionsH-LG V1 V2 V3up to 5 m 5 m 2 m 1 m – Loadup to 5 m 5 m 2 m 1 m – Loadup to 5 m 3 m 1 m 1 m – Loadup to 5 m 5 m 5 m 1 m – Load

Figure 3A

Figure 3B.

Figure 3. µS-VTF device Preparation.

VTF tip is wiped, inserted into a CBS straw, sealed and plunged into LN2.


37

Figure 4A

Figure 4B.


38

Thawing/Warming ProceduresTo initiate a thaw procedure, thaw dishes are first

prepared (Figure 4B). The S3 VTF system uses a 5-step thaw dilution (T1 – T5). The dish can be prepared using a 60 x 15mm culture dish with 25-50 µl droplets (labeled T1 to T5) under oil, or in 6-well dishes using larger volumes (100-200 µl) in air, if desired. A secondary thaw dish (35 x 10mm) should be prepared with 100 µl of the T1 solution. Additionally, a 15 ml of thaw bath medium (1.0M Sucrose in H-LG or H-HTF media; 1.0M Suc = 17.1g Suc/50ml flask) should be warmed to 37°C, in capped culture tubes until needed or directly in a 60 x 15mm dish. Dilution of the thaw bath down to 0.5M Sucrose is an acceptable, practical measure. Next, prepare a half liter, stainless steel dewar flask filled with LN2. Identify the storage tank, canister and cane of the patient sample you wish to thaw. The cane should be isolated from the canister, within the internal neck of the LN2 storage tank. Once grasped, lower the storage canister back to the bottom, while gently raising the Cane ID label to the top of the tank. Confirm ID and quickly remove the cane into the dewar flask, at which point the patient ID on the goblet can be confirmed without additional ambient exposure (Figure 4A). The dual color labeling of the mS-VTF system expedites the identification of the proper specimen in a highly efficient manner.

1) Prepare accessory items needed to effectively thaw a mS-VTF straw, including a sharp, strong surgical scissor (e.g., Mayo scissors), Drummond Micropipet (Fisher Scientific, Microcap bulb assembly Cat#1-000-9000 ), a Stripper pipetter and prepared paperwork.

2) Isolate the straw(s) to be thawed, and return any Cane possessing residual straws back to the LN2 storage tank, again minimizing exposure of the samples to ambient air.

Note: Vitrified specimens, in contrast to frozen samples, are less insulated to brief temperature changes (>5 sec at room temperature could be detrimental). Therefore extreme precautions should be instituted by all lab staff members in their daily use of storage tanks. By storing the mS-VTF straws in intact goblets, which fill with LN2 when submerged, this feature helps insulate the vitrified samples to temperature changes during transitional movements into and out of the primary storage tank or the dewar flask, thus increasing safety time outside LN2.

Top off the dewar flask with LN2, and grasp the isolated straw you desire to thaw using long forceps. Bring the labeled, sealed end above the surface level and confirm the sample ID (Figure 4A). Next, grasp the straw with your scissors just below the inner plug of the CBS™ straw, being careful to keep the inner VTF tip submerged in LN2. Gently tap your scissor against the dewar to dislodge VTF tip from the inner straw wall, in case it was sticking (i.e., QC measure).

3) With your warm, thaw bath dish and the secondary thaw dish in position on the surface (21-24°C) of the stereomicroscope, initiate the thaw (Figure 4B).

a. Pull the isolated straw out of LN2, grasp the base end between your fingers while cutting open the hydrophobic plug end in a horizontal position. Cutting should be performed in front of or on the side of the stereoscope, but below the surface level to avoid any accidentally movement of the cut end (with the internalized label) potentially contaminating or disrupting the dishes.

b. Once cut (3-5 sec into the thaw step), elevate the base end and tip the open end over the thaw bath, allowing the VTF tip to free fall directly into to 37°C solution. This will facilitate rapid warming (approx. 100°C/sec) of the vitrified product. Be careful not to touch the open straw end to the medium, as this will stop the outward progression of the tip. Note, the 37°C temperature may not be critical for an ideal warming rate (the temperature can fall towards room temperature during the warming phase).

c. Within 5-10 sec, grasp and remove the thawed VTF tip. Insert the base-end of the VTF tip into a Drummond™ Micropipet. Note, there is a 1mm hole in the bulb to eliminate positive pressure upon tip insertion. Then, cover the hole and gently squeeze the bulb and slowly extrude the oocytes or embryo(s) into the T1 thaw dish, being careful to avoid air bubble extrusion. Once the oocyte(s)/embryo(s) have been recovered, remove the VTF tip and reinsert it onto a Stripper pipetter. The thaw step can be repeated, immediately, if there is >1 mS-VTF straw to thaw/patient.

4) When all the oocyte(s) or embryo(s) have been recovered, they can be pipette into a clean T1 droplet in the dilution dish (Figure 4B). The T1 step is 3-5 min total (including thaw dilutions), and 5 min each in the T2 to T5 dilutions. All dilutions are performed at room temperature, except T5 which allows for equilibration to


39

37°C before placement into culture dishes for subsequent short-term incubation until the embryo transfer.

5) If assisted hatching is to be performed on thawed BL’s, it should be performed by the T5 step, as the BL’s may completely reexpand in T5, which is easily achieved by laser drilling.

Validation StudiesStudy 1: Frozen 1-cell mouse embryos (Embryo-

Tech) were cultured to expanded/hatching blastocyst (BL) in tri-gas Sanyo mini-incubators for Proficiency Testing purposes. In Expt. 1, 50 good quality BL were randomly assigned to either a slow-freeze control group (Grp 1) or a vitrification treatment (Grp 2). In Grp 1, BL were diluted in 1.5 M glycerol solution for 10 min + 0.2M Sucrose for 5 min before being loaded and sealed in CBS™ straws, and slow cooled from -7°C to -35°C at 0.5°C/min. Embryos were thawed using a standard Irvine Scientific BL Thaw kit. In Grp 2, BL were vitrified and thawed using the Irvine Scientific VTF Cryo and Thaw kits, as described by the manufacturer, and loaded into a mS-VTF device. In Expt. 2, 20 2-cell embryos were vitrified and thawed, being compared to fresh controls. Each straw or flexipipet contained 5 BL/treatment. In the initial studies, mS-VTF thawing of the VTF tip was performed at room temperature, secured to the Stripper pippetter and directly diluted into T1 solutions.

Study 2: Cooling and warming rate curves were validated using micro-thermocouples and a datalogging device.

Study 3: In Expt. 3, BL survivability was reassessed (n=30 BL) using a modified warming step where the tip was immediately placed in warm H-HTF solution for 10 sec upon removal and then reattached to a pipetter to decant embryos and dilute them serially. All embryos were cultured in LG medium + 5% HSA in 25µl microdroplets. In Expt. 4, 100 expanded to hatching blastocysts (BLs) were vitrified using either the µS- or S3-VTF (0.25ml straw) system. S3-treated BLs were handled, diluted and thawed as described by Stachecki et al (2008). BL recovery, survival and continued BL development was assessed at +24 hr.

ResultsStudy 1. No difference was observed in Expt.1,

between re-freeze treatment groups (mS-VTF and cSF), with 17 of 25 BL (68%) re-expanding and continuing

development 24 hr post-thaw. In Expt. 2, 16 to 20 vitrified 2-cells produced ≥ expanded BLs (80%) at +72hr culture, similar to control cultured 2-cells (80% BL).

Study 2.A series of experimental runs evaluating the cooling

and warming conditions of the mS -VTF device revealed interesting, yet, predictable outcomes. Not surprisingly, the outer CBS™straw container cools and warms at a higher rate (1345°C/min and 2364°C/min, respectively) than the inner denuding pipette (i.e., VTF storage device; 1114°C/min and 975 to 1114°C/min, respectively) under room temperature conditions. Furthermore, a classic bi-phasic warming curve was apparent, whether the thawing pipette was inside (in situ) or outside the straw container (warming rate plateau of 118-125°C/min, respectively), due to insulating condensation. Based on the warming curve information, we determined that sustained rapid warming (>1000°C/min ) would be necessary to optimize post-thaw survival by avoiding potentially harmful devitrification (i.e., ice crystal reformation) at the critical temperature range of -80° to -60°C.

Study 3: In Expt. 3., when the VTF tip was directly placed into a 37°C fluid bath, within 3-5 sec, all of the vitrified BLs (n=30; 6 replicates) survived (100%) and most re-expanded following rapid warming procedures.


40

In Expt. 4., 10 replicates of 5 BLs each/treatment revealed no difference in recovery rates (100%) or survival rates (100%) when using S3 vitrified BLs stored in 0.25ml straws or the mS -VTF system. Continued BL development decreased 10-18% overnight, but was not different (P>0.05) between the µS- and conventional straw-VTF systems (90% and 82%, respectively).

DiscussionCommercial influences are pushing VTF devices into

the marketplace and creating a potential QC nightmare in the IVF industry. The inherent design flaws of some devices with regards to secure labeling, long-term storage and sub-optimal recovery rates could present unnecessary and undesirable liability issues on the part of the fertility center thawing oocytes and embryos. Ultimately, the procedural efficacy and success of vitrification is dependent on 3 key concepts in laboratory practice: clarity (organization), consistency (procedural ease and repeatability), and concentration (technical ease and ability to stay on task). The consideration of these the 3 “C’s” to successful VTF is what led to the development the mS-VTF procedure described in this paper.

The mS-VTF device has been systematically validated to be a simple and reliable approach that minimizes intra- and inter-laboratory technical variation, while providing maximum cryosecurity using sterile products. The rapid cooling rate (1114-1345°C/min) of the mS-VTF double container system was sufficient to maintain a vitreous state and optimum cryosurvival when combined with rapid warming (>6000°C/min) of the VTF tips. A recent report by Isachenko et al. (2007) indicated that a double-cut straw container system (CSS-VTF) can reduce the ultra-rapid cooling rate of vitrification up to 25-fold (15000°C/min in open container versus 600°C/min, respectively) without any effect on BL survival. Both the CSS- and mS-VTF systems offer a simple and effective approach to contamination-free cryostorage. Furthermore, the S3 VTF system developed by Stachecki (2008) which represent a macro-VTF approach (cooling rates <1000°C/min) using standard 0.25 ml straws, common to the IVF industry the past 3 decades, has proven to be clinically effective for blastocysts. In our validation studies, the mS-VTF system proved to be highly effective, similar to the established S3 VTF system.

Both VTF systems offer technical simplicity aimed at reducing intra- and inter-laboratory variation, as well other quality control advantages compared to various VTF devices.

Based on the excellent results achieved in the experimental, validation phases testing the mS-VTF procedure, we have initiated IRB-approved clinical studies and application. Hundreds of human oocytes, zygotes, cleaved embryos and blastocysts have been vitrified by the mS-VTF method. The technical efficacy of the system in clinical practice has been confirmed by the 100% recovery rates attained. The effectiveness of the system has been independent of developmental stage, with survival rates ranging from 90 to 100% (Schiewe et al., 2009b). Finally, in clinical application, the mS-VTF device has proven to be successful, as indicated by multiple ongoing pregnancies and live births following oocyte and blastocyst vitrification.

In conclusion, the µS-VTF system has proven to be an effective procedure that may offer “universal” acceptance to alleviate current quality control concerns with the handling, storage and shipment of vitrified oocytes and embryos. This vitrification procedure incorporates two FDA-approved devices to offer a simple, secure and aseptic system for long-term storage of vitrified products without regards to potential cross contamination issues. n

ReferencesFahy GM. The relevance of cryoprotectant “toxicity” to

cryobiology. Cryobiology 1986;23:1-13.Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification

as an approach to cryopreservation. Cryobiology 1984;21:407-26.Isachenko V, Montag M, Isachenko E, Zaeva V,

Krivokharchenko I, Shafei R, van der Ven H Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod. 2005, 20: 492-96.

Isachenko V, Katkov I, Yakovenko S, Lulat A, Ulug, M, Arvas A, Isachenko E. Vitrification of human laser treated blastocysts within cut standard straws (CSS): Novel aseptic packaging and reduced concentrations of cryoprotectants. Cryobiol 2007, 54:305-09.

Kuwayama M. Highly efficient vitrification for the cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 2007;67:73-80.

Kuwayama M, Vatja G, Kato O, Leibo S. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed 2005a;11:300-08.

Kuwayama M, Vatja G, Ieda S, Kato O. Comparison of open and



41

Supplementation of freeze and thaw solutions with a globulin-rich protein source improves

post-thaw survival and implantation of control-rate cryopreserved blastocysts

Joseph M. Kramer, M.Sc.ab

aPh.D. Candidate, Animal Molecular and CellularBiology Graduate Program, Department of Animal

Sciences. bDivision of Reproductive Endocrinology andInfertility, Department of Obstetrics and Gynecology.

University of Florida, Gainesville, Floridaemail: [email protected]

AbstractObjective: To compare the effects of a globulin-rich

protein source versus HSA supplementation in freeze, thaw and post-thaw solutions during control-rate blastocyst cryopreservation.

Design: Murine in vitro embryo freeze-thaw and thaw-survival study. Restrospective study of frozen human blastocyst thaw-survival and impantation rates.

Setting: Academic ART laboratory.Patient(s): Twenty-eight patient couples undergoing

frozen embryo transfer (FET).Intervention(s): Control-rate cryopreservation

of blastocysts using freeze and thaw solutions supplemented with protein serum substitute supplement (SSS; 10 mg/ ml HSA + 2 mg/ml globulins) or human serum albumin (HAS, 10 mg/ml).

Main Outcome Measure(s): Thaw survival, blastocoele re-expansion, clinical pregnancy and implantation rates.

Result(s): Freeze-thaw and post-thaw solutions supplemented with SSS significantly increased

blastocyst re-expansion (P<.05) and total cell number in murine embryos (P=.057). In addition, retrospective analysis of clinical FET cycles found increased post-thaw survival (P<.01), re-expansion (P<.05) and implantation (P<.05) for blastocysts thawed and cultured in solutions supplemented with SSS compared to HSA.

Conclusion(s): The results from this study suggest that supplementation of freeze, thaw and post-thaw solutions with a protein source containing high fractions of globulins, such as SSS, may benefit thaw-survival.

key Word(s): cryopreservation, globulins, HSA, thaw survival, blastocyst

IntroductionFor more than two decades, the inclusion of

globulin-rich protein sources in culture medium has been suggested to benefit in-vitro development of embryos. Initial studies investigating plasma volume extenders containing a mixture of serum albumin and globulins intended for intravenous use, demonstrated high continuing pregnancy rates if included in embryo culture medium (1, 2). Furthermore, these studies attributed the success largely to the high fractions of α- and β-globulins. Results from those early studies led to the development of commercial preparations of globulin-rich protein supplements, such as Synthetic Serum Substitute (SSS; now manufactured under name

Abstract of this study was presented at the Annual Meeting of the American Society for Reproductive Medicine, Atlanta, Georgia, USA, October 17 to 21, 2009.


42

Serum Substitute Supplement, Irvine Scientific)(3) and Serum Protein Substitute (SPS; SAGE Media). These serum substitutes are now formulated to provide a more suitable pH range as well as appropriate osmolarity for inclusion in embryo culture media. Numerous human and animal studies have demonstrated that these commercially available protein supplements promote blastocyst hatching (4-6) and implantation (7-9). While a mechanism for the interactions of globulins with embryos remains to be established, Pool and Martin (2) suggested that the polyhydroxy nature of globulins may influence their physiochemical interactions with water molecules within the culture environment. These interactions may enhance developing embryo viability through physical mechanisms involving altered water dynamics. As such, these interactions may also benefit embryo cryopreservation, where osmoregulation of the embryo is essential for post-thaw re-expansion and survival.

Modern trends seeking to limit the number of freshly transferred embryos along with general improvements

in culture conditions often result in embryos remaining post transfer which places emphasis on successful blastocyst cryopreservation. Previous attempts to improve blastocyst cryopreservation have focused on various components, such as type of cryoprotectant (10, 11), altering start temperatures and rate of slow-freezing (12), utilization of straws verse cryo-vials (13), inclusion of various macromolecules (14) and more recently vitrification (15). One of the earlier reports demonstrating high pregnancy rates from frozen-thawed blastocysts was accomplished with freeze and thaw solutions supplemented with a globulin-rich protein source (16), suggesting globulins may promote post-thaw blastocyst survival. Surprisingly, no studies have evaluated the effects of globulins on blastocyst cryopreservation.

In the present study, assessment of the supplementation of freeze and/or thaw solutions with SSS compared to HSA alone for control-rate cryopreservation of murine blastocysts in an effort to determine if globulin-rich protein sources offer advantages during cryopreservation. We hypothesize that freeze and thaw solutions supplemented with a globulin-rich protein, such as SSS, will improve thaw-survival, as measured by blastocyst re-expansion and total cell number. In addition, we retrospectively examined human blastocysts thawed and cultured in solutions supplemented with SSS compared to HSA alone to determine if globulin-rich protein supplementation benefited clinical frozen embryo transfer (FET) outcomes.

Materials and MethodsFreezing and Thawing of BlastocystsFreeze and thaw procedures followed a modified

version of Menezo’s protocols (17, 18) using kits of commercially available solutions (G-FreezeKit Blast and G-ThawKit Blast; Vitrolife, Englewood, CO). Each freeze and thaw solution was supplemented with protein in the form of HSA (10 mg/ml albumin; Vitrolife) or SSS (10 mg/ml albumin + 2 mg/ml globulins; Irvine Scientific,Santa Ana, CA) according to the experimental design. Unless noted, all procedures were carried out at 37°C. For freezing, blastocysts were exposed for 5 min to a MOPS buffered incubation medium containing no cryoprotectant or sucrose. They were then transferred

F.O.B. Westbury, NY. Frozen material will be shipped with dry ice. For research only. Not for human or diagnostic use. For in-vitro cell culture only.

Chick Embryo Extract (CEE)

Available From Our

Westbury, NY Inventory

Chick Embryo ExtractFROZEN

Chick Embryo ExtractLYOPHILIZED

Product #CE650T Product #CE650TL

1 x 20ml 1 x 10ml

10 x 20ml 10 x 10ml

1.516.333.22211.800.645.62641.516.997.4948 1.800.255.9378

www.accuratechemical.com

ACCURATE CHEMICAL & SCIENTIFIC CORPORATIONIT’S MORE THAN A NAME - IT’S A PROMISE

Westbury, NY

FAXWest Coast Office

©20

03 A

ccu

rate

Ch

emic

al &

Sci

enti

fic C

orp

.

9467 Chick Embryo Ad 7/1/05 1:27 PM Page 1


43

into a solution of 5% glycerol + 0.1 M sucrose for 10 min, and then to a solution of 10% glycerol + 0.2 M sucrose for 7 min. Blastocysts were loaded into CBS high security straws (Cryo Bio Systems, IMV corp, New York, NY), sealed and transferred into a programmable rate controlled freezer (Cryologic, Victoria, AU). The freezing program initially cooled from 18°C to -7°C at a rate of -2°C/min. Ice crystallization seeding was performed manually by touching the straw with pre-cooled forceps. The temperature was held for 15 min and then further cooled at a rate of -0.3°C/min to -38°C. Straws were then plunged directly into LN2 (-196°C) for storage. For thawing, straws were removed from LN2, held in air at room temperature for 1 min, and immersed into a water bath for 30 sec at 37°C. Straws were wiped to remove excess water and then cut to expel the contents into a petri dish containing 10% glycerol + 0.2 M sucrose. Once located, the blastocysts were transferred into a solution of 5% glycerol + 0.1 M sucrose for 5 min, then into a solution of 0.1 M sucrose for 5 min, then held in MOPS buffered incubation medium for 5 min.

Animal Experimental DesignFrozen two-cell murine embryos (Embryotech,

Haverhill, MA) were thawed and cultured in G1v5 PLUS (Vitrolife) containing 5mg/ml HSA for 72 h. Only resulting expanded blastocysts, containing a discernable inner cell mass and a well defined layer of trophoblast cells were cryopreserved. Blastocysts remained in liquid nitrogen storage for at least 24 h but less than 1 week. Following thaw, blastocysts were cultured for 24 h and then assessed for thaw survival. Blastocysts exhibiting greater than 50% blastocoele re-expansion were considered to survive.

Experiment 1: Effects of SSS Supplemented Freeze and Thaw Solutions on Post-Thaw Murine Blastocyst Re-Expansion

Murine blastocysts were randomly distributed to one of four treatments: frozen and thawed in cryopreservation solutions supplemented with HSA (group I), frozen in solutions supplemented with HSA and thawed in solutions supplemented with SSS (group II), frozen in solutions supplemented with SSS and thawed in solutions supplemented with HSA (group III),

and frozen and thawed in solutions supplemented with SSS (group IV). Following thaw, each treatment was cultured for 24 hrs in G2v5 PLUS (Vitrolife) containing HSA (5 mg/ml).

Experiment 2: Effects of post-thaw culture in medium containing SSS on re-expansion of murine blastocysts having been frozen-thawed with different protein supplements

Murine blastocysts were randomly distributed to one of four treatments as described in experiment 1. Following thaw, each treatment was cultured for 24 hrs in G2v5 supplemented with SSS (5 mg/ml albumin + 1 mg/ml globulins).

Experiment 3: Effects of protein supplementation in freeze-thaw and post-thaw culture solutions on murine blastocyst re-expansion and total cell number

Murine blastocysts were randomly distributed to freeze-thaw solutions supplemented with HSA followed by 24 hrs culture in G2v5 PLUS containing HSA (5

Manuscript Request

The Journal of Clinical Embryology™ is now

requesting the submission of original data

manuscripts for peer review. Please send

your contribution by email, in Word format,

to:

[email protected]

Our Journal is registered with the

Library of Congress, ISSN: 1941-1901


44

mg/ml) (group I) or freeze and thaw in solutions supplemented with SSS followed by 24 hrs culture in G2v5 supplemented with SSS (5 mg/ml albumin + 1 mg/ml globulins) (group II). Total cell numbers were calculated from all blastocysts that re-expanded using a simple fixation and staining procedure. Briefly, individual blastocysts were exposed for 2 min in hypotonic solution [1.0% sodium citrate (Sigma-Aldrich, St. Louis, MO) + 3 mg/ml BSA (Sigma-Aldrich)], then transferred into a softening solution [0.01N HCl (Sigma-Aldrich) + 1% tween 20 (Sigma-Aldrich)] for 10 sec and loaded onto a slide containing 10 ul softening solution. As soon as the softening solution evaporated, one drop of fixative solution (methanol:acetic acid; 3:1) was added to fixate nuclei to slide. The central location on the slide containing the nuclei was encircled with a carbide pen. Coverslips were mounted with 10 ul of vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI)(Vector Laboratories, Burlingame, CA) applied to the slide area containing nuclei. The total number of nuclei corresponding with each blastocyst were counted using a Nikon Eclipse E600 (Nikon, Melville, NY) epifluorescent microscope equipped with a 60X dry objective.

Human Retrospective AnalysisA retrospective study was conducted that included

28 patient couples that initiated FET cycles from January 2006 to March 2009 at Shands hospital at the University of Florida, Gainesville, Florida. Only patients who had frozen blastocysts thawed for the purpose of FET were included. No other exclusion criteria were used. All women were pre-treated by combined oral contraceptive pills followed by down-regulation with daily leuprolide acetate. Following hormonal suppression, confirmed by estradiol levels (<40 pg/ml) and transvaginal ultrasound assessment of ovaries and the endometrial stripe, patients were placed on transdermal estradiol with incresing incremental doses in order to develop endometrium. Once the endometrial thickness reached 7mm, progesterone in oil was injected IM to synchronize endometrium for embryo transfer. On day 5 of progesterone administration, blastocysts, originally frozen on developmental day 5 or 6 with at least a grade of 3BB or better using Gardner’s scoring system (19) were thawed. One to two blastocysts were transferred to each patient on day 6 of initiation of progesterone treatment regardless of the developmental stage of the blastocyst. Pregnancy was first determined 9 days post transfer with a serum hCG greater than 25

Treatment Thawed-Recovered Re-Expanded (%)Group I: HSA-->HSA 75 53 (70.7)ab

Group II: HSA-->SSS 74 60 (81.1)ac

Group III: SSS-->HSA 75 43 (57.3)b

Group IV: SSS-->SSS 76 68 (89.5)c

Treatment Thawed-Recovered Re-Expanded (%)Group I: HSA-->HSA 38 36 (94.7)Group II: HSA-->SSS 40 37 (92.5)Group III: SSS-->HSA 41 37 (90.2)Group IV: SSS-->SSS 39 37 (94.9)

Table 1. Effects of protein supplement in freeze-thaw solutions on murine post-thaw re-expansion.

Table 2. Effects of post-thaw culture medium with SSS on re-expansion of murine blastocyst frozen-thawed with different protein supplements.

Results with different superscripts indicate significance. ab vs c P<0.05, b vs. ac P<0.01, b vs. c P<0.001.

No significance observed between treatments.


45

mIU/ml. Ongoing clinical pregnancy was determined 4 weeks post transfer using transvaginal ultrasound to assess the presence of fetal heartbeat.

We compared two groups of patients. Group I (n=19) included patients who had frozen blastocysts thawed and cultured in solutions supplemented with HSA. Group II (n=9) included patients who had frozen blastocysts thawed and cultured in solutions supplemented with SSS. Thaw-survival was characterized as blastocysts exhibiting fewer than 20% degenerative cells assessed at two hrs post-thaw. Blastocyst expansion was assessed at 24 hrs post-thaw with only blastocysts exhibiting greater than 50% blastocoele expansion considered to have re-expanded. The institutional review board and ethics committee of the University of Florida approved this study.

Statistical AnalysisAll data were analyzed using software SPSS

statistics 17.0 (SPSS Inc., Chicago, IL). Chi-Square testing was used to analyze blastocyst re-expansion for animal experimental studies 1, 2, and 3, as well as the clinical retrospective study. Multiple comparisons with Bonferroni adjustments were used to compare individual treatments in animal experimental studies 1 and 2. Student’s t-test was used to compare mean total cell numbers for animal experiment 3. Fisher’s exact test was used to analyze clinical pregnancy and implantation rates for the retrospective study. Significance was deemed P<.05.

ResultsEffects of Supplementing Freeze and/or Thaw

Solutions with SSS on Post-Thaw Murine Blastocyst Re-Expansion

Initially 306 murine blastocysts were frozen using freeze solutions supplemented with either HSA (n=152)

or SSS (n=154). Table 1 shows the distribution of 300 recovered murine blastocysts following thaw. Overall, there was no significant difference in re-expansion from blastocysts frozen in HSA compared to SSS, whereas re-expansion was greater if blastocysts were thawed in solutions supplemented with SSS (85.3%) compared to HSA (64.0%) (P<.05). Individually, 89.5% of recovered blastocysts re-expanded from group IV compared to 70.7% and 57.3% in groups I (P<.05) and III (P<0.001), respectively.

Effects of post-thaw culture medium with SSS on re-expansion of murine blastocyst frozen-thawed with different protein supplements

In experiment 2, 158 of 160 frozen murine blastocysts were recovered following thaw and were grouped similarly to experiment 1 (Table 2). Following After 24 h of culture in post-thaw medium supplemented with SSS, blastocyst re-expansion was excellent in all groups with no significant differences between treatments.

Effects of protein supplementation of freeze-thaw and post-thaw culture solutions on murine blastocyst re-expansion and total cell number

Experiment 3 included 111 murine blastocysts frozen, thawed and cultured for 24 h using cryosolutions and post-thaw culture medium supplemented with HSA or SSS (Table 3). A greater percentage of blastocysts re-expanded if frozen, thawed and cultured in solutions supplemented with SSS (85.5%) compared to HSA alone (66.1%; P<.05). In addition, the mean total cell number of re-expanded blastocysts, as determined by staining of fixed nuclei with DAPI (Figure 1), was moderately greater for SSS group compared to HSA group (80.0 vs 70.5; P=.057).

Table 3. Effects of protein supplementation of freeze-thaw and post-thaw culture solutions on murine blastocyst re-expansion and total cell number.

Treatment Thawed Expanded (%) Mean Total Cell Count

SEM

Group I: HSA 56 37 (66.1)a 70.5c 4.03Group II: SSS 55 47 (85.5)b 80.0c 2.96

Treatments within column with different superscripts are significantly different. a vs. b P<0.05, c vs. c P = 0.057.


46

Figure 1. Epifluorescence imaging of total cell number froma frozen-thawed murine blastocyst fixed and stained with DAPI. The total number of nuclei corresponding with each blastocyst were counted at 600X total magnification.

Retrospective Analysis of Frozen-Thawed Human Blastocyst Re-Expansion Using Thaw and Post-Thaw Solutions Supplemented with SSS

The retrospective analysis included 82 blastocysts originally frozen in freeze solutions supplemented with HSA (Table 4). Cycles were grouped based on the thaw protocol in use at the time. Between January 2006 and June 2008, 58 embryos were thawed using thaw and post-thaw culture medium supplemented with HSA (group I). Beginning in July 2008 through March 2009, 24 additional blastocysts were thawed, but in these cases using thaw and post-thaw culture medium supplemented with SSS (group II). Blastocysts thawed and cultured in solutions supplemented with SSS had significantly greater survival at 2 hrs post-thaw and greater blastocyst re-expansion at 24 hrs than blastocysts thawed and cultured in HSA alone (83.3% and 66.7% vs 43.9% and 15.8%, respectively; P<.01). In addition, there was a higher clinical

Table 4. Retrospective Analysis of Frozen-Thawed Human Blastocyst Re-Expansion Using Thaw and Post-Thaw Solutions Supplemented with SSS

Group I Group II Statistical Significance

Number of Cycles 19 9Number of Embryos Thawed 58 24Average Number of Embryos Thawed 3.1 2.7 NSNumber of Embryos Recovered (%) 57/58 (98.3) 24/24 (100.0) NSNumber of Embryos Survived (%) 25/57 (43.9) 20/24 (83.3) P<0.01Number of Embryos Expanded (%) 9/57 (15.8) 16/24 (66.7) P<0.01Number of Embryo Transfers (%) 11/19 (57.9) 8/9 (88.9) NSAverage Number of Embryos Transferred 1.4 1.6 NSNumber of Biochemical Pregnancy Per Thaw (%)Per Transfer (%)

44/19 (21.1)4/11 (36.4)

55/9 (55.6)5/8 (62.5)

NSNS

Number of Clinical PregnancyPer Thaw (%)Per Transfer (%)

22/19 (10.5)2/11 (18.2)

55/9 (55.6) 5/8 (62.5)

P<0.05NS

Number of ImplantsPer Embryo Transferred (%)

22/15 (13.3)

77/13 (53.8) P<0.05


47

pregnancy rate per thaw and higher implantation rate from frozen blastocysts thawed and cultured in solutions containing SSS compared to HSA (55.6% and 53.8% vs 10.5% and 13.3%; P<.05).

DiscussionThe aim of this work was to improve current freezing

and thawing protocols in order to enhance post-thaw survival and implantation of blastocysts. The present study was designed to test whether inclusion of the commercially available globulin-rich protein supplement SSS in freeze, thaw and post-thaw solutions would increase thaw-survival as compared to HSA. The results indicate that the type of protein supplement provided impacted thaw survival, as measured by post-thaw re-expansion and total cell number. As several studies have demonstrated post-thaw re-expansion and total cell number are predictors of implantation, the selection of protein type used in freeze, thaw and post-thaw solutions could affect frozen embryo transfer outcomes.

Accumulated evidence supports a role for globular protein supplementation in embryo culture medium. Results from the current study suggest globulin-rich protein sources, such as SSS, may also promote thaw-survival of cryopreserved blastocysts. The results show that blastocysts cryopreserved and thawed in freeze-thaw solutions using SSS had significantly higher rates of re-expansion compared to HSA alone. If we consider that cellular mechanisms involved during blastocyst expansion are likely to also be involved in re-expansion of collapsed blastocysts following thaw, then these results are consistent with previous studies demonstrating the positive effects of globulin-rich protein supplements on blastocyst hatching (4-6). Interestingly, transitioning from SSS used during freeze to HSA used during thaw negatively affected post-thaw re-expansion. Schneider and Hayslip (4) observed a similar phenomena during extended embryo culture where compact embryos cultured with a mix of HSA and globulin proteins resulted in poor production of hatched blastocysts when moved to a sequential medium devoid of globulins, suggesting an active role for globulins in stimulation of blastocyst hatching. In addition, the present study found that the culture

of thawed blastocysts overnight in SSS promotes excellent blastocyst hatching, regardless of protein type in freeze-thaw solutions. These results imply supplementation of post-thaw culture medium with a globulin-rich protein source, like SSS, may restore the potential for blastocyst re-expansion even if previously frozen and/or thawed with HSA.

Total cell numbers were also analyzed to determine if re-expanded blastocysts from SSS treatments were generally healthier compared to HSA alone. As total blastocyst cell number has been correlated with implantation potential (20), our results suggest that blastocysts cryopreserved and thawed in solutions containing SSS are more robust than blastocysts cryopreserved and thawed in HSA. Although murine blastocysts were not transferred to recipients, similar studies assessing protein supplementation in routine embryo culture found globulins enhanced total cell numbers (21) and implantation (7-9), further supporting the impression that blastocysts cryopreserved, thawed and cultured in solutions containing SSS are more likely to implant.

Based on the research results using murine embryos, our clinical ART laboratory, which previously solely used HSA, incorporated SSS into freeze-thaw protocols as well as post-thaw culture of human blastocysts. Since this change, our program has shown a remarkable improvement in thaw-survival. In addition, as our standard procedure is to culture thawed embryos overnight, we have observed significant improvements in blastocyst re-expansion prior to transfer. These changes have led to significant increases in pregnancy per thaw and implantation rates. However, as these numbers are small, additional studies are needed to determine whether these trends are clinically significant.

Given the osmotic dynamics imposed on embryos undergoing exposure to high concentrations of cryoprotectants, minor alterations in the physiochemical environment may dramatically enhance an embryo’s ability to restore osmotic function. Pool and Martin (2) propose that the high number and orientation of hydroxyl groups (-OH) in polyhydroxy compounds may interact with water molecules affecting physical properties such as exchange dynamics. The finding


48

presented here that thawed blastocysts are more likely to re-expand if cultured with a protein source containing globulins lends support to the idea that polyhydroxy compounds improve osmotic function. Intriguingly, other polyhydroxy compounds, some of which are currently used in IVF medium, may function better than globulins for cryopreservation. Hyaluronan, which is naturally found in the reproductive tract (22) and has been reported to stimulate in vitro embryo development and improve implantation (23), was previously shown to produce equally high thaw-survival rates in mouse and cow embryos when substituted for newborn calf serum in freezing solutions (24). While no study has evaluated the affects of hyaluronan in freeze-thaw solutions of human blastocysts, a recent study comparing blastocyst FET using hyaluronan-rich transfer medium, embryoglue (VitroLife), demonstrated no improvement in implantation as observed with fresh blastocyst transfer (25). While it remains to be elucidated, Korosec

(25) suggested that the freeze-thaw process itself may affect receptor-mediated actions of hyaluronan thereby disrupting signaling between the endometrium and the embryo. As this could have major implications affecting implantation, further research is needed.

Alternative mechanisms for the thaw-survival promoting effects of globulins are plausible and warrant further investigation. A previous in vitro culture study of murine embryos cultured in medium supplemented with SSS showed reduced presence of reactive oxygen species (ROS) accompanied by lower incidences of apoptosis when compared to embryos cultured in medium supplemented solely with HSA (5). Our study was limited to observational data, including blastocyst re-expansion and total cell number. We did not analyze post-thaw culture medium for ROS or re-expanded blastocysts for apoptosis and therefore cannot exclude this as a potential mechanism promoting thaw-survival. In addition, globulin proteins within SSS have also been suggested to act as energy substrates or serve as carriers of growth promoting factors. While this is plausible, it is more likely that the –OH groups within polyhydroxy compounds, like SSS, influence osmotic tolerance of embryos. In support, a recently made available supplement containing a mixture of HSA and dextran (DSS, Irvine Scientific) is being promoted to be as effective as SSS for in vitro mouse embryo development and more relevantly produce comparable thaw-survival to SSS when used as the protein component in vitrification solutions for all stages of development. As dextran is a nonproteinaceous polymer known to interact strongly with water, future studies incorporating DSS may provide additional support for a mechanism in which polyhydroxyl compounds interact in a physiochemical manner with the embryo and its environment to enhance osmoregulation rather than function as growth promoting factors.

The results of this study suggest that supplementation of freeze, thaw and post-thaw solutions with a protein source containing high fractions of globulins, such as SSS, may benefit thaw-survival. As this study was limited to slow-cool cryopreservation, it would be interesting to see if similar results can be obtained through vitrification. Given that most referenced


49

vitrification solutions are formulated using SSS, it is likely that the success of those solutions may at least in part be due to the presence of globins.

AcknowledgmentsThe author graciously acknowledges the

contributions of Larissa Ali for technical assistance in the laboratory, Dr. Kenneth Drury for critically reviewing this manuscript, and the physicians and nurses of the University of Florida Reproductive Medicine group for assistance with the retrospective clinical study. This study was presented in part at the Annual Meeting of the American Society for Reproductive Medicine, Atlanta, Georgia, USA, October 17 to 21, 2009. n

References1. Adler A, Reing AM, Bedford JM, Alikani M, Cohen J.

Plasmanate as a medium supplement for in vitro fertilization. J Assist Reprod Genet 1993;10:67-71.

2. Pool TB, Martin JE. High continuing pregnancy rates after in vitro fertilization-embryo transfer using medium supplemented with a plasma protein fraction containing alpha- and beta-globulins. Fertil Steril 1994;61:714-9.

3. Weathersbee PS, Pool TB, Ord T. Synthetic serum substitute (SSS): a globulin-enriched protein supplement for human embryo culture. J Assist Reprod Genet 1995;12:354-60.

4. Schneider EG, Hayslip CC. Globulin-enriched protein supplements shorten the pre-compaction mitotic interval and promote hatching of murine embryos. Am J Reprod Immunol 1996;36:101-6.

5. Esfandiari N, Falcone T, Agarwal A, Attaran M, Nelson DR, Sharma RK. Protein supplementation and the incidence of apoptosis and oxidative stress in mouse embryos. Obstet Gynecol 2005;105:653-60.

6. Tucker KE, Hurst BS, Guadagnoli S, Dymecki C, Mendelsberg B, Awoniyi CA et al. Evaluation of synthetic serum substitute versus serum as protein supplementation for mouse and human embryo culture. J Assist Reprod Genet 1996;13:32-7.

7. Desai NN, Sheean LA, Martin D, Gindlesperger V, Austin CM, Lisbonna H et al. Clinical experience with synthetic serum substitute as a protein supplement in IVF culture media: a retrospective study. J Assist Reprod Genet 1996;13:23-31.

8. Dugan KJ, Shalika S, Smith RD, Padilla SL. Comparison of synthetic serum substitute and fetal cord serum as media supplements for in vitro fertilization: a prospective, randomized study. Fertil Steril 1997;67:166-8.

9. Ben-Yosef D, Yovel I, Schwartz T, Azem F, Lessing JB, Amit A. Increasing synthetic serum substitute (SSS) concentrations in P1 glucose/phosphate-free medium improves implantation rate: a

comparative study. J Assist Reprod Genet 2001;18:588-92.10. Li R, Trounson A. Rapid freezing of the mouse blastocyst:

effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen. Reprod Fertil Dev 1991;3:175-83.

11. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Englert Y. Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum Reprod 2000;15:905-10.

12. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril 2003;79:407-10.

13. Van Den Abbeel E, Van Steirteghem A. Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability. Hum Reprod 2000;15:373-8.

14. Kuleshova LL, Shaw JM, Trounson AO. Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology 2001;43:21-31.

15. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril 2006;86:20-6.

16. Veeck LL, Bodine R, Clarke RN, Berrios R, Libraro J, Moschini RM et al. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil Steril 2004;82:1418-27.

17. Menezo YJ, Veiga A. Cryopreservation of blastocysts. In: Gomel V, Leung PCK, eds. In Vitro Fertilization and Assisted Reproduction. Vancouver: Monduzzi, 1997:49-53.

18. Menezo YJ. Blastocyst freezing. Eur J Obstet Gynecol Reprod Biol 2004;115 Suppl 1:S12-5.

19. Gardner DK, Schoolcraft WB. In vitro culture of human blastocysts. In: Jansen R, Mortimer D, eds. Towards reproductive certainty: fertility and genetics beyound 1999. Carnforth, UK: Parthenon Publishing, 1999:378-88.

20. Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil 1997;109:153-64.

21. Tanikawa M, Harada T, Ito M, Yoshida S, Iwabe T, Terakawa N. Globulins in protein supplements promote the development of preimplantation embryos. J Assist Reprod Genet 1999;16:555-7.

22. Yaegashi N, Fujita N, Yajima A, Nakamura M. Menstrual cycle dependent expression of CD44 in normal human endometrium. Hum Pathol 1995;26:862-5.

23. Gardner DK, Rodriegez-Martinez H, Lane M. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum Reprod 1999;14:2575-80.

24. Palasz A, Alkemade S, Mapletoft RJ. The use of sodium hyaluronate in freezing media for bovine and murine embryos. Cryobiology 1993;30:172-8.

25. Korosec S, Virant-Klun I, Tomazevic T, Zech NH, Meden-Vrtovec H. Single fresh and frozen-thawed blastocyst transfer using hyaluronan-rich transfer medium. Reprod Biomed Online 2007;15:701-7.


50

vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres. 2004 Theriogenology 61:1125-1135

Harvey AJ, Kind KL, Pantaleon M, Armstrong DT, Thompson JG. Oxygen-regulated gene expression in bovine blastocysts. 2004 Biol Reprod 71:1108-1119

Booth PJ, Holm P, Callesen H. The effect of oxygen tension on porcine embryonic development is dependent on embryo type. 2005 Theriogenology 63:2040-2052

Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner DK. A proteomic analysis of mammalian preimplantation embryonic development. 2005 Reproduction 130:899-905

Kind KL, Collett RA, Harvey AJ, Thompson JG. Oxygen-regulated expression of GLUT-1, GLUT-3, and VEGF in the mouse blastocyst. 2005 Mol Reprod Dev 70:37-44

Feil D, Lane M, Roberts CT, Kelley RL, Edwards LJ, Thompson JG, Kind KL. Effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. 2006 J Physiol 572:87-96

Rho GJ, S B, Kim DS, Son WJ, Cho SR, Kim JG, B MK, Choe SY. Influence of in vitro oxygen concentrations on preimplantation embryo development, gene expression and production of Hanwoo calves following embryo transfer.2007 Mol Reprod Dev 74:486-496

Kubisch HM, Johnson KM. The effects of blastomere biopsy and oxygen tension on bovine embryo development, rate of apoptosis and interferon-tau secretion. 2007 Reprod Domest Anim 4:509-515

Balasubramanian S, Son WJ, Kumar BM, Ock SA, Yoo JG, Im GS, Choe SY, Rho GJ. Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos. 2007 Theriogenology 15:265-275

Leoni GG, Rosati I, Succu S, Bogliolo L, Bebbere D, Berlinguer F, Ledda S, Naitana S. A low oxygen atmosphere during IVF accelerates the kinetic of formation of in vitro produced ovine blastocysts. 2007 Reprod Domest Anim 42:299-304

Mechanical aspects of using low oxygen:Fukuda M, Fukuda K, Ranoux C. Unexpected low oxygen tension

of intravaginal culture. 1996 Hum Reprod 11:1293-1295Avery B, Melsted JK, Greve T. A novel approach for in vitro

production of bovine embryos: use of the Oxoid atmosphere generating system. 2000 Theriogenology 54:1259-68

Allen CB, Schneider BK, White CW. Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia. 2001 Am J Physiol Lung Cell Mol Physiol 281:L1021-L1027

Conaghan J, Steel T. Real-time pH profiling of IVF culture medium using an incubator device with continuous monitoring 2008 J Clinical Embryology 11(Summer):25-26

Lighting in the laboratory:Noda Y, Goto Y, Umaoka Y, Shiotani M, Nakayama T, Mori T.

Culture of human embryos in alpha modification of Eagle’s medium under low oxygen tension and low illumination. 1994 Fertil Steril 62:1022-1027

Nakayama T, Noda Y, Goto Y, Mori T. Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos. 1994 Theriogenology 41:499-510

Ottosen LD, Hindkjaer J, Ingerslev J. Light exposure of the ovum and preimplantation embryo during ART procedures. 2007 J Assist

Reprod Genet 24:99-103Takenaka M, Horiuchi T, Yanagimachi R. Effects of light on

development of mammalian zygotes. 2007 Proc Natl Acad Sci 4:14289-14293

Oh SJ, Gong SP, Lee ST, Lee EJ, Lim JM. Light intensity and wavelength during embryo manipulation are important factors for maintaining viability of preimplantation embryos in vitro. 2007 Fertil Steril 88(suppl 4):1150-1157

Mineral oil:Provo MB, Herr C. Washed paraffin oil becomes toxic to mouse

embryos upon exposure to sunlight. 1998 Theriogenology 49:214Otsuki J, Nagai Y, Chiba K. Peroxidation of mineral oil used

in droplet culture is detrimental to fertilization and embryo development. 2007 Fertil Steril 88:741-743

Otsuki J, Nagai Y, Chiba K. Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture. 2009 Fert Steril 91:1745-1749

Sifer C, Pont J-C, Porcher R, Martin-Pont B, Benzacken B, Wolf J-P. A prospective randomized study to compare four different mineral oils used to culture human embryos in IVF/ICSI treatments. 2009 Eur J Obstet Gyn Reprod Biol 147:52-56

Khan Z, Morbeck DE, Walker DL, Barnidge DE. Washing mineral oil reduces but does not eliminate a known contaminant. 2009 Fertil Steril 92:S233

Hughes PM, Morbeck DE, Hudson SBA, Fredrickson JR, Walker DL, Coddington CC. Peroxides in mineral oil used for in vitro fertilization: defining limits of standard quality control assays. 2010 J Assist Reprod Genet DOI: 10.1007/s10815-009-9383-x

Zone(s) of physicochemistry:Stokes PJ, Abeydeera LR, Leese HJ. Development of porcine

embryos in vivo and in vitro; evidence for embryo ‘cross talk’ in vitro. 2005 Dev Biol. 284:62-71

Gopichandran N, Leese HJ. The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos. 2006 Reproduction 131:269-77

Buffers:Swain JE, Pool TB. New pH-buffering system for media utilized

during gamete and embryo manipulations for assisted reproduction. 2009 Reprod Biomed Online 18:799-810

Quiet Embryo Hypothesis:Leese HJ. Quiet please, do not disturb: a hypothesis of embryo

metabolism and viability. 2002 Bioessays 24:845-849Leese HJ. What does an embryo need? 2003 Hum Fertil 180-185Leese HJ, Sturmey RG, Baumann CG, McEvoy TG.Embryo

viability and metabolism: obeying the quiet rules. 2007 Hum Reprod 22:3047-3050

Baumann CG, Morris DG, Sreenan JM, Leese HJ. The quiet embryo hypothesis: molecular characteristics favoring viability. 2007 Mol Reprod Dev 74:1345-1353

Leese HJ, Baumann CG, Brison DR, McEvoy TG, Sturmey RG. Metabolism of the viable mammalian embryo: quietness revisited. 2008 Mol Hum Reprod 14:667-672

Sturmey RG, Hawkhead JA, Barker EA, Leese HJ. DNA damage and metabolic activity in the preimplantation embryo. 2009 Hum Reprod 24:81-91

Techniques to achieve low oxygen tension for culture of human embryos - References (continued from page 32)


51

closed methods for vitrification of human embryos and elimination of potential contamination. Reprod Biomed 2005b;11:608-14.

Liebermann J, Tucker MJ. Vitrifying and warming of vitrified human oocytes, embryos and blastocysts: Vitrification procedures as an alternative to conventional cryopreservation. Methods Mol Biol 2004, 254:3435-64.

Rall W. Avoidance of microbial cross-contamination of cryopreserved gametes, embryos, cells

and tissues during storage in liquid nitrogen. Embryologists Newsletter 2003; 6(2):1-7.

Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196oC by vitrification. Nature 1985;313:573-75.

Rall WF, Wood MJ, Kirby C, Whittingham DG. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987;80:499-504.

Reed ML. Shipping vitrified embryos – Should we be concerned about exposing very small volume specimens to nitrogen vapor temperatures and brief ambient conditions? Clin Embyrologist 2007; 10:19-30.

Schiewe MC. Comparative studies of estrous synchronization, ovulation induction, luteal function and embryo cryopreservation in domestic sheep and application to related investigations of nondomestic ungulate species. Ph.D. Dissertation,1989, pp. 102-124.

Schiewe MC, Rall WF, Stuart LD, Wildt DE. Ovine embryo cryopreservation: Analysis of cryoprotectant, cooling rate and in situ straw dilution using conventional freezing or vitrification. Theriogenology 1991;36:279-93.

Schiewe, MC and RE Anderson. A novel approach to embryo vitrification by LN2 vapor storage of stripper tips® in CBS™ high security straws. Proc. Ann. Pacific Coast Reprod. Soc. Mtg., Fertil Steril (Suppl 2), 89:S23, 2008.

Schiewe MC and GM Fahy. Validation of a simple and effective sterile vitrification system for embryos. Proc. 64th Ann. Amer. Soc. Reprod. Med. Mtg., Fertil Steril (Suppl 1), 90: S288, 2008.

Schiewe, MC, N Nugent, K Crawford and S Zozula. MicroSecure vitrification (µS-VTF) of mouse blastocysts: comparison to S3 vitrification using 0.25 ml straws or Cryopettes®. Proceeding of AAB Conference, June 2009a:35-36.

Schiewe, MC, GM Fahy and RE Anderson. MicroSecure vitrification (µS-VTF): simple, safe, sterile, secure and clinically successful. Proc. 65th Ann. Amer. Soc. Reprod. Med. Mtg., Fertil Steril (Suppl 1), 92: S___, 2009b.

Stachecki JJ, Garrisi J, Sabino S, Caetano JPJ, Wiemer K, Cohen J. A new safe, simple, and successful vitrification method for bovine and human blastocysts. Reprod Biomed Online 2008; 17:360-67.

Vajta G, Nagy Z. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. RBM Online 20068;12:779-96.

Vanderzwalmen PV, Bertin G, Debauche Ch, Standaer V, Bollen N, van Roosendal E, Vandervorst M, Schoysman R, Zech N Vitrification of human blastocysts with the hemi-straw carrier: application of assisted hatching after thawing. Hum Reprod 2003, 18:1504-1511.

EQUIPMENT1. Tri-gas/Temperature Controlled Incubators a. MCO-5M, Sanyo2. Surface warmer/37oC heating block (Labline) 3. Stereomicroscope 4. Pipetters: Pipetman or Eppendorf (P-20, P-200),

Stripper™, and Drummond™ Micropipet* *Order part as: Bulb Assembly for Microcaps (Fisher Scientific #13681451)

5. LN2 storage tank(s) (MVE or Taylor-Warton)6. Dewar flask, stainless steel (≥0.5L)7. Syms® Automatic Sealer8. Forcep and Dissecting scissors (Mayo and Delicate)

DISPOSABLE SUPPLIES1. Culture Dishes (Falcon or Nunc) a. 100 X 30mm (F-1003) b. 60 X 15 mm (F-3652) c. 35 X 10mm (F-3001)2. Cultureware (Falcon; Mid-Atlantic Diagnostics)a. 17 X 100mm r.b.culture tubes, snap cap, 14 ml (F-

2057), 20/sterile packb. 50 ml Flask, non-vented (F-3014)3. Disposable Pipettes (Falcon; Mid-Atlantic

Diagnostics)a. 1 ml (F-7521); IND, ST, 100/box, 1000/csb. 5 ml (F7543); IND, ST, 25/pk, 200/csc. 10 ml (F-7551); IND, ST, 25/pk, 200/cs4. Pipetters: Pipetman or Eppendorf (P-20, P-200);

Stipper™; Drummond™ Micropipet5. Denuding Pipettes (Cook Medical); 300µm ID, 10/

tube, sterile6. Yellow pipette tips (Eppendorf ), 1000/case7. HSW syringes, 30ml , ind. sterile pkg (Biogenics)8. Acrodisco.22µm filters, #SLG VR25LS (Millipore

Corp.), 100/box

CRYO SUPPLIES1. 0.3 ml CBSTM embryo straws, ind. pkg, sterile (Irvine

Scientific)2. Colored ID Rods, CBSTM

3. Colored labeling tape, ¾” wide4. Sharpie marker, fine tip

MEDIA PRODUCTS1. Culture Medium a. Hepes buffered –Global® Medium or a.a. enriched

Human Tubal Fluid, 100 ml b. Vitrification Media (S3, or comparable VTF solution)c. Thawing Media Kit (T1-T5)1. Sucrose, #S1888 (Sigma Chemical Co.)

MicroSecure Vitrification (µS-VTF) Procedure - References (continued from page 40)

©2010 Irvine Scientific P/N 10328R Rev.0

For fertilization through blastocyst development; Allowing you to stock and order one medium leading to time and cost savings while providing a nurturing environment for your precious embryos.

Single Step Medium™

For more information on this product or to obtain a sample please contact your territory manager or call 1-800-437-5706.

Now available with a new and improved formulation

10328R Clinical Embryologis Ad -SSM Rev0.indd 1 4/15/2010 4:52:22 PM

Clinical E The Journal ofmbryology … · combines the beneﬁ ts of a precurved guiding catheter...

Documents

Transcript of Clinical E The Journal ofmbryology … · combines the beneﬁ ts of a precurved guiding catheter...