The Journal of
Clinical Embryology Volume 12, Issue 1 • Spring, 2009
ISSN 1941-1901
6 weeks
© Lennart Nilsson
The Official Journal of The College of Reproductive Biology - American Association of Bioanalysts. w w w.embr yolog i st s .com
PRSRT STD US POSTAGE PAID PERMIT 3933 FORT LAUD FL
Š COOK 2009
Providing nutritional balance from gamete to embryo. WH-BA-CM-EN-200902
The Cook Medical sequential culture media system is a family of products formulated to contribute the appropriate nutrients to match the shifting metabolic requirements of gametes and embryos. The system includes solutions for gamete selection and preparation that optimize fertilization rates, cleavage solutions that build embryo viability for transfer, and highnutrient solutions that produce quality blastocysts. To learn more about our full line of ART materials and devices, contact your Cook Medical representatives or visit us at www.cookmedical.com.
Embryo Culture S E Q U E N T I A L C U LT U R E M E D I A S Y S T E M
AO RT I C I N T E RVE N T I ON
CA R DI OLOGY
CR I TI CA L CA R E
END OSCOPY
PERIPHERAL INTERVENTION
SURGERY
UROLOGY
WOME N ’ S H E ALT H
The Journal of Clinical Embryology™
TABLE OF CONTENTS Editor’s Corner: “Embryospeak”........................... 3
Volume 12, Issue 1
Editor’s Corner: “EmbryoSpeak” Singleton – Twins – Triplets
Sextuplets – Septuplets -
Ken Drury, Ph.D
OCTUPLETS!
Letters to the JCE Editor........... 7 Assisted hatching as a technique for use in human in vitro fertilization and embryo transfer is long overdue for careful and appropriate study....9 Michael J Tucker and G David Ball
Anti-Mullerian Hormone and the Embryologist............... 15 William E. Roudebush and Jessica M. Mattke
Northern California Association for Reproductive Biologists (NCARB) and Bay Area Reproductive Endocrinologist Society (BARES) Joint Annual Meeting 10-9-08........................ 25 A comparison of day-3 versus day-2 and day-5 versus day-4 embryo transfers among in-vitro fertilization patients.... 27
Taguchi Sagiri, M.D.; Funabiki Miyako, M.D.; Ujino Hiroaki, M.D.; Hayashi Terumi, M.D.; El Beltagy Khalid, M.D.; Amano Namiko, M.T.; Nakamura Yoshitaka, M.D.
Meetings for the Embryologist............................. 35 Featured Websites.................... 40 JCE Editorial Board and New Member Welcome.................... 41
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.
– Quadruplets – Quintuplets -
Ken Drury, Ph.D., Editor
H
igher Order Multiples (HOM) is a term used to denote pregnancies brought about by the multiple implantation of embryos which in many cases have been conceived through the use of one type or another of Assisted Reproductive Technologies (ART). These pregnancies, more often than not, lead to premature births resulting in morbid and/or deadly outcomes. A working definition of HOM is generally accepted as an ongoing gestation comprised of three or more fetuses, whether or not all are viable and able to survive to term. The public has been awed in the past by sensational reports of HOM births stemming from either natural means (Dionne quintuplets born May 28, 1934 - the first quintuplets known to survive infancy and the only all female identical set of five ever) or more recently through fertility drug treatment (sextuplet babies born to the Hayes family in 2004; as well as, the McCaughey septuplets - born November 19, 1997) These reported events brought generally sympathetic, if not congratulatory responses, from the public at large. Unfortunately, in the case of the Dionne Quints, these children wound up as wards of the State and later as a circus “side-show”. So, what are we to make of the latest media-storm over the birth of Octuplets in California? Is there any current accurate information available to help us assess this phenomenal event? The only thing certain at the present time is that there are eight premature sibling infants residing in a California NICU unit with a mix of six brothers and sisters awaiting them in a mortgage threatened house surrounded by a firestorm of media attention. (Four of the eight infants were released from hospital at time of press. -Ed.) Of course, we as reproductive specialists are stunned by this shocking news and need to understand in more detail (as soon as possible) what the actual facts surrounding the use of ART treatment were in this particular case. We must also begin to assess the impact of this event on our own infertility practices and what it may bring to our profession. In particular, how should laboratory personnel (from directors, supervisors, technologists and technicians) respond to the news that six embryos (Frozen or Fresh?) were loaded into a catheter and transferred to the uterus of a 34 year old women who had already demonstrated (superb) fecundity on several previous occasions? I will return to this question in just a bit. However, although this remains a valid question, we have to ask ourselves if the above event actually took place in the manner it has been reported?
3
The Journal of Clinical Embryology™
Volume 12, Issue 1
The infertility clinic reported to have provided the ART expertise in this occasion was headed by a non-specialized OBGYN physician who was also grand-fathered into the position of laboratory director. The SART success rate for this program, reported for the year 2007, was the worst of any of the approximately 400 plus clinics in America (3/37 = 8% live births per cycle). It is all the more astonishing since this program’s implantation rate was an anemic 3% even when transferring on average 4 embryos per case in this patient’s age bracket (<35). The fact that the worst cases of HOM births taking place within the last two decades did not originate from IVF or the transfer of embryos, and that it has not been
confirmed that there are actually identical twins involved in the birth of these (Octuplet) infants, raises the question of how exactly were these babies conceived. It has been suggested in the blog section of the Los Angeles Times, http://latimesblogs.latimes.com/ lanow/2009/02/the-american-so.html, (Medical group examining Beverly Hills fertility doctor in octuplets case by Shelby Grad 3:52 PM | February 10, 2009 http:// latimesblogs.latimes.com/lanow/2009/02/the-american-so.html) that this physician, while trying to raise his overall ART success rates, simply used artificial insemination to produce this Octuplet pregnancy. Is it unreasonable to think that Ms. Seulman would not understand the difference between hormonally stimulated intrauterine insemination (IUI) and IVF, especially if she were told it was IVF. Below is a more cynical perspective offered by one of the article’s blog responders: “Let’s get real. Octomom has not shown herself to be honest or trustworthy. False and misleading statements,hiding information, excess cash for spending on luxury items (plastic surgery, fake nails, hotel room). Multiple aliases and heavy use of welfare and disability for herself (disability from a government hospital job) and for kids (for ADHD?) She is clearly not one to shy from any scam. This guy, the doctor, probably did not implant a single embryo into Suleman. The eight kids are most likely the result of using too much fertility drugs on a ‘patient’ who is relatively young and fertile. Most likely he paid her to be an advertisement for his otherwise very unsuccessful fertility clinic. And got on television in 2006 doing just that. Come on, you guys are reporters, not transcribers. The real question is who is the father. Dr. Kamrava? Grandfather Ed (Adnan?) Daoud (David?) Suleman (Soloman?)(yuck) Or someone else? The taxpayer needs to know so he can contribute to the cost of these kids.” Posted by: CalvinB | February 13, 2009 at 11:40 AM All this aside, it is certainly “conceivable” that this Octuplet incident did take place in the manner described by most news broadcasts; that is, by using IVF and the transfer of “extreme” numbers of embryos. If this is the case, what responsibilities do IVF laboratory personnel share with any infertility physician performing embryo transfers? Within our professional societies, there are published guidelines issued (American Society of Reproductive Medicine (ASRM); Society of Assisted Reproductive Technology (SART)) which present reasonably clear
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 #4 PO Box 650790 Vero Beach, FL 32965-0790 Phone 352.331.5235 Fax 352.331.6250 E-mail: embryospeak@bellsouth.net www.embryologists.com
4
The Journal of Clinical Embryology™
Volume 12, Issue 1
scenarios and suggestions pertaining to the transfer of embryos (Fertil Steril 90:S163–164 2008). Other important reports on this subject have come from the respected national organization Resolve (also see: Blastocyst culture and transfer in clinical-assisted reproductioninfertility treatment and multiplegestation pregnancy, A Publication of RESOLVE:The National Infertility Association http://www.resolve.org/site/DocServer/Multiple-Gestation-Pregnancy.pdf?docID=621). At the same time, individual IVF-ET programs should evaluate their own data to identify patient-specific, embryo-specific, and cycle-specific determinants that may influence the likelihood of implantation and live birth during a particular treatment case. This is important in order to develop embryo transfer policies that minimize the occurrence of HOM gestations. Resolve’s Practice Committee Opinion also reviews the published literature relating to the potential benefits, pitfalls, and risks of blastocyst culture. These are not laws, but they do make a strong case for the curtailment of HOM pregnancies, especially when the question addresses how many embryos should be considered for transfer. Laboratory personnel need to be well informed about the contents and rational contained in these particular guidelines and be able and willing to share them with the transferring physician if the need arises. Knowledge of current pertinent literature can also have important benefits as illustrated in a study presented in Human Reproduction by Roberto Matorras et al. (2005, 20#10:29232931) entitled: The implantation of every embryo facilitates the chances of the remaining embryos to implant in an IVF programme: a mathematical model to predict pregnancy and multiple pregnancy rates. Within this article, the authors state “We recommend using the aforementioned formula to quantify the pregnancy rate and the risk of multiple pregnancy in the counseling of the infertile couple at embryo transfer. Such a formula is freely available at www.ifca.unican.es/matorras/mathpreg/ “. Individual infertility programs likewise should discuss situations or conditions which may arise that could influence or alter the implementation of these guidelines. In general, consideration should be given to the transfer of fewer blastocyst stage embryos than cleavage stage embryos, particularly in women with excellent prognoses
and high-quality blastocysts (Guidelines for the number of embryos to transfer following in vitro fertilization No. 182, September 2006 in Int J Gynaecol Obstet. 2008 Aug;102(2):203-16). Why is it important for laboratory personnel to be up to date and knowledgeable about not only the technical aspects of gamete preparation, embryo culture, and proper transfer techniques but also the medical and ethical implications of infertility treatment? It is because every member of the laboratory (Director, Supervisor, Technologist, Technician and Trainee) is a key individual of the overall patient treatment team. Each provides expertise in critical areas of laboratory function responsible for the production of viable embryos, as well as, important information concerning specific characteristics of embryo development taking place during a particular procedure. To put this in perspective, here is a statement taken from a website instructing infertility patients about issues surrounding the embryo transfer procedure: “Transferring Embryos to the Uterus - Embryos are transferred on either day three or day five of development. The embryologists at GRS are highly-skilled in identifying “healthy” embryos and in some cases will recommend that a patient extend embryo development to day five, known as the blastocyst stage. Blastocyst transfer has become quite common in IVF cycles as it can increase chances for success while decreasing the likelihood of multiples. Your physician will work closely with the embryologists to determine if a day three or day five transfer would be ideal for your cycle. The transfer of several embryos increases the probability of success. A multiple embryo transfer also increases the risk of a multiple pregnancy. Any multiple pregnancy carries an increased risk of miscarriage(s), premature labor and premature birth as well as an increased financial and emotional cost. Pregnancy-induced high blood pressure and diabetes are more common in women pregnant with more than one fetus. Prolonged hospitalization may be necessary for these pregnant women and for the mother and babies after delivery. Tubal (ectopic) pregnancy is also possible, and a combination of normal pregnancy and ectopic pregnancy may occur. A tubal pregnancy is a condition that may require laparoscopy or major surgery for treatment.” (Georgia Reproductive Specialists - http://www.ivf.com/overview.html).
5
The Journal of Clinical Embryology™
Volume 12, Issue 1
This is heavy responsibility indeed, and the lives of patients and resulting offspring can, in many cases, be directly connected to the IVF Laboratory. Let there be a prominent notice placed in your laboratory that reads: “Our Laboratory Embryologists work closely with your physician to bring about the best and healthiest infertility treatment outcome possible”. This means providing your physician with the laboratory skills and ethical commitment that go hand in hand with producing happy healthy families. Remember, the time of transfer is not the ideal occasion to have patients considering for the first time how many embryos to have transferred. Patients need to be well aware of your program’s policy concerning the number of embryos generally considered for transfer and how these policies may relate to them specifically. Every program should also have an infertility psychologist available to counsel patients concerning this most important aspect of their treatment. All of these considerations will impact the quality of your program as well as the overall field of infertility treatment. Before leaving you, please check the “Letters to the JCE Editor” section and begin to formulate your own questions and comments to the articles you read here in JCE. Also, I welcome a new member to our JCE Editorial Board: Dr. Alan Thornhill, Ph.D. HCLD from The London Bridge Fertility Centre in London England. Dr. Thornhill is the first board member residing outside the United States and we look forward to Alan’s contributions to our Journal. Please check out his Bio and contact information in this issue and on the JCE Website: www.embryologists.com. n
Sad News I am sorry to report that Rev. Dr. Timothy Appleton passed away on Saturday, March 14, 2009 in a Cambridge Hospital after a 6 month battle with inflammation of the pancreas. His family, wife Jan and sons Jon and Simon, have arranged for burial on Tuesday, March 31st, 2009. A public service will be held at 2 pm at the Parish Church of Harlton, Cambridgeshire (UK) followed by a private cremation. Tim held two doctorates in Cell Biology and was an ordained Anglican Priest - in Non Stipendary capacity. His interest since 1982 have been as an Independent Fertility Counsellor working with licensed IVF Centres in the UK. For 23 years he was senior counsellor at Bourn Hall Clinic - founded by the pioneers of IVF, Patrick Steptoe & Robert Edwards. A close friendship connected him with Patrick Steptoe. His role has been to try and help and support patients suffering from infertility and undergoing treatment, whether they achieved their goals of having a family or not. Tim’s recent writing was to help parents and children understand the miracle of IVF. — Fred Zander
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 jceadvertising@bellsouth.net or 352-331-5235. Visit today and save our website to your favorites.
6
The Journal of Clinical Embryology™
Volume 12, Issue 1
Letters to the JCE Editor (Ed Note: JCE readers are encouraged to express their views, experience and expertise in this column. It is not necessary to respond to a published article in order to make your viewpoints known) To the Editor:
used to perform the procedures. The hallmark of good science is to be able to have other labs or groups test your methods and theories to see if they are reproducible. That apparently can not be done in this case. I’m not saying the article should be retracted, only that the authors should provide a more complete description of their methods in the next edition.
I’m writing in regards to the article authored by Drs. Stachecki and Cohen (S3 Vitrification System: A Novel Approach To Blastocyst Freezing) published in the winter 2008 issue (Vol.11(4):5-14) of the Journal of Clinical Embryology. I found it interesting (as I suspect many others did) that there may be a good way to vitrify blastocysts within standard freezing straws, so I was interested in trying to perform this particular method in my own laboratory. However, when looking through the article, I found no description of the solutions used; only a reference to an article in Reprod Biomed Online in 2008 (A new safe, simple and successful vitrification method for bovine and human blastocysts. Sep;17(3):360-7). When I looked at that article, there again was no mention of how the freeze and thaw solutions were prepared.
Jeff Boldt, Ph.D. HCLD JBoldt@ecommunity.com
Author Response:
We have given our contact info in the article. If anyone would like to try out our method they simply need to contact us. There are many labs now testing our method. And, I may add, proving to themselves that our theories and methods are sound. I might also add that the clinical data in the article is not from our lab, rather it comes from labs who are using the method we developed and have graciously shared their data and experience with us. We invite other labs to try our method if they are unsatisfied with their current freezing method.
My question to you is whether this article should have been published without exact details of the methods
Manuscript Request
James J. Stachecki, Ph.D. Cryobiologist Tyho-Galileo 3 Regent Street, Suite 301 Livingston, NJ 07039 973-436-5037 james@galileoivf.com
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:
embryospeak@bellsouth.net
Comment and Communication in response to:
Our Journal is registered with the
Venkat G, Thornhill A, Wensvoort S & Craft I. Does laser assisted hatching using partial zona thinning (LAH) improve outcome in frozen embryo transfer (FET) cycles? J Clin Embryol 2008;11:17-30.
Library of Congress ISSN: 1941-1901
(continued page 9)
7
Life’s beginning in safe han
Life’s beginning in safe hands
www.medicult.com
Somewhere around the world a baby is born with the help of MediCult media every 15 minutes. That’s impressive in any language.
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
Assisted hatching as a technique for use in human in vitro fertilization and embryo transfer is long overdue for careful and appropriate study Michael J Tucker* and G David Ball Shady Grove Fertility RSC, Rockville, MD 20850, USA (MJT); Seattle Reproductive Medicine, Seattle WA 98109 (GDB). *Correspondence: michael.tucker@integramed.com
A
ssisted hatching (AH) as an adjunct to IVF-ET has been around for a long time (Cohen et al 1990); and as Venkat et al (2008) rightly pointed out in their recent paper, it has only been minimally studied, especially in a randomized manner. While their efforts to raise consciousness on this subject are appreciated by ourselves, we were disappointed that their own study was merely a retrospective review of five year old data, which ultimately just suggested inconclusively that AH was not beneficial following embryo thawing and transfer (FET). We felt it worth responding to this paper both to point out some shortcomings of the Venkat study; and to make some proposals moving forward, with the hope that we might stimulate more definitive study of this subject. We would initially like to make it clear that we are advocates of the use of laser systems for zona pellucida (ZP) ablation, in particular for embryo biopsy procedures where this hands-free technology enables a much faster, and more convenient approach to opening the ZP than either chemical, or mechanical means (Han et al 2003). We suspect that with regard to this procedure that the laser has become the tool of choice. On the other hand, assisted hatching, irrespective of the technical approach, remains a procedure of largely unproven worth regardless of how theoretically appealing it might seem to apply to poorer prognosis IVF-ET cases. The temptation to apply AH to repeat failure patients that may well include older patients, those with elevated basal FSH, cases of consistently poor embryo morphology and the like, is very real. But in so doing we continue to operate less as a science and more as a
therapeutic art! That said, we have attempted to dispel some of these possible myths. For example, it is doubtful that an elevated basal FSH by itself is sufficient grounds to warrant application of AH (Assemi et al 2006); but although this was analyzed retrospectively in this particular study, it does, nevertheless, prompt future study of this preliminary conclusion in our clinics. There does exist a little more circumstantial evidence to justify application of AH to poorer quality embryos. Morphologically challenged embryos consistently develop more slowly in vitro (Tucker & Liebermann 2003). Also in a non-randomized, but controlled study of the application of AH to fresh blastocyst transfers, it was shown that blastocysts that formed on day-6 benefited from AH compared to control unmolested blastocysts (Tucker 1999). Further, in vitro observation of over 300 research embryos left in culture up till day-9 of development showed a distinct reduction in both blastocyst formation, and hatching relative to increased age of the patient from whom the embryos were donated (Porter et al 2002). Additionally, hatching occurred less often the more slowly the embryo took to reach the blastocyst stage. Obviously a major caveat of this study is that it was an in vitro observational analysis, rather than a true in vivo implantation study; but it does profoundly implicate the role of a compromised ZP to restrict blastocyst hatching both in older women, and in couples whose embryos develop more slowly. This ties in well with the aforementioned study of the benefit of AH for more slowly developing blastocysts (Tucker 1999). Consequently, we routinely apply AH using the laser in women 38yrs of age and older, and those consenting couples that present with poorly developing embryos.
9
The Journal of Clinical Embryology™
Volume 12, Issue 1
Clearly both of these patient groups might benefit from better prospectively randomized controlled study of the impact of AH. So why is this not done? One cynical stance is that if AH were to prove ineffectual, then this would mean that a fee generating procedure would be dropped from the IVF laboratory. Another reason is that undertaking such a well-controlled study can be very painstaking. For example, in the case of a extremely well-undertaken study of AH it may take a very long time to recruit sufficient patients, as was the case where AH was applied to good prognosis IVF patients which took over three and a half years to complete (Sagoskin et al 2007). On the face of it, this study was a waste of time, because no benefit was observed from the use of AH. The converse of this is that it was good science reporting a null outcome, the real benefit of which was to indicate that if AH was carried out appropriately, then it was in no way deleterious to the embryos to which it was applied, nor to their eventual ability to implant. And therein lies the rub… Applying AH appropriately is the key point here; and we would like to comment on the differing technologies available in passing, but more specifically the most user-friendly laser systems that can be utilized. While acidified Tyrode’s medium became for a while these authors’ approach of choice to ablate the ZP both for embryo biopsy and AH, we have since changed to the more convenient and quicker laser for both procedures, due to clear benefits in terms of embryonic development post-biopsy when the latter was applied versus the acidified medium approach (Han et al 2003). Mechanical opening of the ZP is possible, and avoids the problems encountered with potentially lingering chemicals, but technically it can be demanding, and inconsistent unless undertaken by very skilled hands. Mechanical dissection of the ZP is also not sufficiently subtle to enable partial ablation of the ZP, unlike the acidified medium approach or the laser. This brings us to our first concern with the Venkat paper which applied a large area ZP thinning approach to AH, which in our opinion is not effective (e.g., Tucker et al 1993) for AH, and more worrisomely may in fact be harmful to the embryo. When it comes to ZP ablation, it is important to be cognizant of the potential harm that any approach to AH might be imposing on the embryo be it mechanical, chemical, or in this case using the laser. Not all
lasers that have been developed for micromanipulation of the human embryo are equal. The greatest danger of pointing a laser in the general direction of an embryo is thermal damage. Therefore to know the thermal characteristics of a laser beam is essential to minimize any potential harm. Lasers of wavelength 1480–1450nm are used for penetration of the embryo’s ZP; and laser pulses of duration 100µsec to 20millisec have been used. Powers of 300mW and 47mW have been reported with different lasers. If we compare the heating in neighboring tissue caused by the laser pulse, this enables us to estimate potential thermal damage to the embryo caused by the laser. The theory of pulsed heating has been described in detail (Douglas-Hamilton & Conia 2001; Tadir & Douglas-Hamilton 2007), and can be used to compute the maximum temperature reached at any given distance from the laser beam axis. An equivalent way of expressing this is to calculate the radius at which a given maximum temperature is reached during the pulse. The circle at that radius describes the isothermal region, or isotherm for short, corresponding to that temperature. If we look at the two laser systems as applied in two different studies, we can get a better idea how each laser system might have very different effects on the clinical outcomes. In the case of the system used by Sagoskin et al (2007), the laser power was 300mW with a pulse-duration of 500µsec [ZILOS-tk]. In the case of the Venkat study, the laser power was 47mW with a pulse-duration of 20millisec [Fertilase]. Therefore it can be seen for the above two laser pulses applied to the embryos the following isotherm radii, in microns, were generated:
Isotherm Temperature
Laser Laser 300mW 47mW Pulse 500µsec Pulse 20msec
140°C 100 80 60 50 40
7.8µm 10.5 12.6 16.1 19.3 27.5
0µm 1.2 14.6 36.6 56.7 108.0 (continued page 13)
10
Zander Scientific, Inc. 772-569-5955 • www.zandair.com THE ULTIMATE Medical Laboratory PURIFICATION SYSTEM WINNER OF THE 2005 AHR INNOVATION AWARD HVAC - In-duct Photo-Catalytic Air Purifier delivers total air purification to every laboratory room
ZAND-AIR 150/200 PCOC™ PhotoCatalytic Oxidation Chamber
ZAND-AIR 150/200 GS™ “Green Series” PhotoCatalytic Air Purification ZAND 200GS eries Green S 4 to 5 acity Tons Cap
ZAND 150GS
MIX
eries Gree1n S 3 /2 to 1 acity Tons Cap
and/or
MATCH
ZAND-AIR 150GS™ and ZAND-AIR200GS™ are encased units with filters installed and can be fitted into ducting to provide an airtight flow of the airstream through these units.
ZAND-AIR 150PCOC™ and ZAND-AIR 200PCOC™ are not encased and need to be installed airtight inside HVAC systems in the plenum of the AHU upstream of the fan unit.
zIVF-AIRe 100C™
FDA 510(k) # KO41952 DESIGNED FOR USE IN IVF LABORATORIES FRONT POSITION: Activated Carbon Filter with specially formulated gas adsorption media (including zeolite and potassium permanganate) adsorbs automobile exhaust fumes, organic hydrocarbons, formaldehyde from particle boards used in construction, paint, solvents, chlorine, cleaning chemicals, volatile organic compounds (VOCs), chemically active compounds (CACs) and other harmful agents. BACK POSITION: Hospital Grade HEPA Filter individually tested by the supplier and certified to remove particles of 0.3 µm in an approved aerosol with not less than 99.97% efficiency. Garment particles, skin flakes, pollen, mold, fungal spores, dust mites, and bacteria are examples of micro particles. PHOTO-CATALYTIC OXIDATION attacks the molecular bond of VOCs and CACs, viruses and bacteria and renders them harmless.
England, Ireland, Scotland & Wales contact:
Atlas Environments Ltd. Tel: +44 161-221-9655 www.atlasenv.com
“Science
Asia, Middle East and Europe (except England, Ireland, Scotland & Wales) contact: MTG – Medical Technology Vertriebs-GmbH Tel: +49 871-97519-0 www.mtg-de.com
is organized knowledge.”
~ Herbert Spencer
Subscription
FORM
.com
www.embryologists The Journal of
Clinical Embryology
PLEASE FAX COMPLETED FORM TO US +1-772-569-9667
DIRECTIONS:
Simply fill out this form in it’s entirety and fax to our office. +1-772-569-9667 United States: Mexico and Canada: • $30.00 (4 issues) Outside North America: • $40.00 (4 issues)
Subscriber Details:
Subscriber Name: _________________________ Subscriber Email: _________________________ Company/Institute: ________________________
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 EXPRESS 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.
Thank you for
SUBSCRIBING... Medical Market Consultants, Inc.
Tel: +1-352-331-5235 Fax: +1-772-569-9667 Email: jceadvertising@bellsouth.net Web site: www.embryologists.com
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
(continued from page 10) These two differing cases are shown in sketches below (Figures 1. & 2.). The embryo is estimated to be 120Âľm in diameter, and the isotherms are scaled accordingly. The contents of the embryo are not shown, but clearly one would like to disturb them thermally as little as possible.
While we are grateful to Venkat et al for reporting their outcomes from the long term use of AH as they applied it to thawed embryos, we would like to question not only the validity of their results, but in addition wanted to point out the potential harm that they were applying to the thawed embryos for the duration of their study. We routinely apply AH with the Zilos-tk laser (more recently using just a 300microsec) to all of our thawed embryos. This is most commonly applied to cryopreserved blastocysts (e.g., Liebermann & Tucker 2006), as they pass through the sucrose dilution steps, during which time the blastocyst is usually collapsed. For AH of fresh day-6 blastocysts, we expose the embryo briefly (~1min) to 0.2M sucrose solution to collapse it partially, which allows safe laser ablation of the ZP at a site away from the inner cell mass (Sagoskin et al 2002). While we feel that this technology as we apply it for AH is at worst simply benign, it is essential that we undertake future studies to see if it is truly beneficial in the patients where we think it is helpful. This seems precisely to be the message that has been sent out by the SART/ASRM practice committees (2008)
Fig. 1 Fertilase: Power 47mW, pulse duration 20millisec
Fig. 2 ZILOS-tk Power 300mW, pulse duration 500microsec
DISTANCE LEARNING Jones Institute for Reproductive Medicine Eastern Virginia Medical School Norfolk, Virginia
Although the drilled-hole diameter is comparable, the volume heated in the interior of the embryo is much higher, and the heating lasts 40 times longer in the case of the Fertilase system. From this simple representation it is easy to conclude that when the Fertilase system is applied, even if not for full opening of the ZP, nevertheless the embryo may be in significant danger of thermal shock. This would be further amplified in an approach such as used by Venkat et al (2008), where 25% of the entire ZP was thinned extensively.
Now accepting applications for Spring 2010 Application Deadline is December 1, 2009
13
The Journal of Clinical Embryology™
Volume 12, Issue 1
with reference to the use of AH, where it is not seen to be universally beneficial, but perhaps clinically useful in welldefined patient sub-groups. It remains for us to define such sub-groups more effectively. n
hatching in in vitro fertilization: A review of the literature. A committee opinion. Fertil Steril 2008;90:S196-8. Sagoskin AW, Han T, Graham JR, Levy MJ, Stillman RJ, Tucker MJ. Healthy twin delivery after day 7 blastocyst transfer coupled with assisted hatching. Fertil Steril 2002;77:615-7. Sagoskin AW, Levy MJ, Tucker MJ, Richter KS & Widra EA. Laser assisted hatching in good prognosis patients undergoing in vitro fertilization-embryo transfer: a randomized controlled trial. Fertil Steril 2007;87:283–7. Tadir Y & Douglas-Hamilton DH. Laser effects in the manipulation of human eggs and embryos for in vitro fertilization. Methods in Cell Biol 2007;82:409-31. Tucker M. Relevance of assisted hatching with blastocyst stage transfer. Proceedings First World Congress on Controversies in Obstetrics, Gynecology & Infertility, Prague, Czech Republic, Oct 28-31, 1999; Monduzzi, Bologna, Italy, pp49-52. Tucker MJ, Luecke NM, Wiker SR & Wright G. Chemical removal of the outside of the zona pellucida of day-three human embryos has no impact on implantation rate. J Assist Reprod Genetics 1993;10:187-91. Tucker MJ & Liebermann J. Morphological scoring of human embryos and its relevance to blastocyst transfer. In: Patrizio P, Tucker MJ, Guelman V, eds. A Color Atlas for Human Assisted Reproduction. Philadelphia, PA: Lippincott, Williams & Wilkins, 2003:99-108. Venkat G, Thornhill A, Wensvoort S & Craft I. Does laser assisted hatching using partial zona thinning (LAH) improve outcome in frozen embryo transfer (FET) cycles? J Clin Embryol 2008;11:17-30.
References:
Assemi A, Richter KS, Tucker MJ, Sagoskin AW. Does assisted hatching benefit patients with an elevated basal follicle stimulating hormone (FSH)? Fertil Steril 2006;86:2S; P-363, ppS333. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP & Weimer K. Impairment of the hatching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Hum Reprod 1990;5:7-13. Douglas-Hamilton DH & Conia J. Thermal effects in laser-assisted pre-embryo zona drilling. J Biomedical Optics 2001;6:205-13. Han T, Graham J, Tucker MJ, Davis A, Sagoskin AW. Laserassisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis: two successful case reports. Fertil Steril 2003;80:453-5. 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. Porter R, Tucker M, Graham J & Sills ES. Advanced embryo development observed during extended in vitro culture: observations of formation and hatching patterns in non-transferred human blastocysts. Human Fertility 2002;5:215-20. The Practice Committees of SART & ASRM. The role of assisted
SOME EMBRYOS NEED EXTRA ATTENTION Fairfax Cryobank Stores Infectious Reproductive Specimens Fairfax Cryobank has over 20 years experience storing infectious and potentially infectious reproductive specimens. Let us store your quarantine specimens, having confidence that their storage will be handled professionally and with full compliance to regulatory requirements.
Your Embryo Storage Solution 1(866) 442-0408 • Fairfaxcryobank.com Fairfax, VA • Austin, TX • Philadelphia, PA
14
� Quarantine storage tanks to accommodate embryos, sperm, and oocytes. � Use of own shipper tanks for safer transfer. � Specimens stored in LN2 vapor tanks. � Tanks are monitored 24/7. � Storage available in Fairfax,VA, Austin, TX, and Philadelphia, PA. � FDA compliant and licensed by NY, CA, and MD. � Most economic fees.
The Journal of Clinical Embryology™
Volume 12, Issue 1
Anti-Mullerian Hormone and the Embryologist William E. Roudebush and Jessica M. Mattke Beckman Coulter, Inc., Chaska, MN, USA Corresponding Author: William E. Roudebush, PhD, Beckman Coulter, Inc., 1000 Lake Hazeltine Dr., M/S R-280C, Chaska, MN 55318-1084; Phone: 952-368-1124; eMail: WERoudebush@Beckman.com Introduction
warranted as a direct determinant of ovarian status, as compared to day 3 FSH. This review examines the potential of AMH to facilitate assisted reproductive technologies.
T
he primary function of the female ovary is the production of a mature and viable oocyte capable of fertilization, subsequent embryo development and implantation. At birth, the ovary contains a finite number of oocytes available for folliculogenesis. This finite number of available oocytes is termed the “ovarian reserve.” The determination of ovarian reserve is important in the assessment and treatment of infertility. As the ovary ages, the ovarian reserve diminishes. The peak reproductive capacity for women is obtained prior to the age of 30 years (Faddy and Gosden, 1996). Ovarian reserve also refers to the number and quality of oocytes available to produce a dominant follicle late in the follicular phase of the menstrual cycle at any given age. By women continuing to delay the birth of their first child, the incidence of age related infertility has increased (Baird et al., 2005). A prediction of the likely success of assisted reproductive techniques (ART), such as in vitro fertilization, is possible using better estimates of the ovarian reserve (Freour et al., 2006). Infertility is experienced by nearly one out of every ten couples worldwide who attempt conception during a single year of attempts (Boivin et al., 2007). Currently, the most commonly used biomarker test to assess ovarian reserve is the measurement of menstrual cycle day 3 follicle stimulating hormone (FSH), which is an indirect measurement. Cycle day 3 is chosen since the level of estradiol level is expected to be low and this low level of estradiol in turn affects FSH levels via negative feedback control. However, anti-mullerian hormone (AMH) is another biomarker of ovarian reserve that has been the subject of recent research. AMH offers the potential to provide a more direct determination of ovarian status and does not appear to be affected by menstrual cycle day. Further research into anti-mullerian hormone is
Overview of Reproductive Endocrinology
From the hypothalamus, gonadotropin releasing hormone (GnRH) acts upon the anterior pituitary to produce both FSH and luteinizing hormone (LH), both of which target the female ovary. FSH is responsible for follicular recruitment, growth and estrogen (mostly β-estradiol) conversion from androgens during folliculogenesis. Estrogens (e.g. estradiol, E2) are the primary hormones that provide negative feedback to the hypothalamus and anterior pituitary to inhibit FSH and LH secretion. LH is responsible for final follicular and oocyte maturation, subsequent ovulation and corpus luteum (CL) formation. During folliculogenesis, LH acts upon the ovarian theca cells to produce androgens. Following ovulation, LH will promote estrogen and progesterone secretion by the CL. In addition to steroidal hormones, the ovary (i.e., granulosa cells) also produces a number of peptide hormones which are members of the transforming growth factor (TGF)-β/activin superfamily. These peptides hormones include relaxin, inhibin A, inhibin B, activin, follistatin and anti-mullerian hormone (AMH): also called Mullerian inhibiting substance (MIS). The actions of activin include granulosa cell proliferation, FSH and LH receptor expression, enhancement of aromatase activity and subsequent estradiol production, suppression of androgen production, inhibins and follistatin production and the enhancement of oocyte developmental competence. The primary action of follistatin is to bind to and neutralize activin. AMH is also produced by the Sertoli cells of the testis
15
The Journal of Clinical Embryology™
Volume 12, Issue 1
Biomarkers: Endocrine Testing
in the male, and exclusively by ovarian granulosa cells in the adult female. During embryonic development in males, secretion of AMH from testicular Sertoli cells is essential for the regression of the Mullerian ducts, and thus the normal development of the male reproductive tract. The Mullerian ducts are the primordium for the uterus, fallopian tubes and upper vagina in the female. In the male, secretion of AMH by the Sertoli cells commences during embryogenesis and continues throughout life. Levels drop following puberty, decreasing slowly to a relatively low post-puberty level. In the female, serum AMH is undetectable until the onset of puberty. AMH is produced in a wide range of follicles from primary to early antral stages of folliculogenesis. The role of AMH in the cycling female is to inhibit follicle recruitment for folliculogenesis, primarily by inhibiting the action of FSH on follicle growth and selection. AMH levels are maintained at relatively constant levels until menopause, at which time AMH falls to undetectable levels.
Biomarkers are desirable for assessing fertility because of the minimal invasiveness of blood collection in comparison to other procedures, reflection of internal function, speed of analysis and reasonable cost. The “magic bullet” biomarker for ovarian reserve has yet to be clearly defined. However many biomarkers correlate to ovarian reserve status. With the implementation of better testing methods and discovery of new biomarkers, more options should be available. There are a few newly recognized biomarkers that look extremely promising, including AMH and inhibin-B. Inhibin B and AMH are examples of direct measures of ovarian reserve, since these hormones are produced during specific stages of follicular development rather than by follicular stimulation. These two biomarkers have recently been receiving additional visibility with the development of more robust and reliable laboratory testing methods. These developments increase the utility of these biomarker assays for clinical research use. In addition to indirect and direct measurements, there are stimulation tests available, which are frequently used to ascertain or estimate ovarian reserve. These stimulation tests require drug exposure, baseline measurements and follow-up measurements of the biomarkers, such as FSH.
Antral Follicle Count Measurements
The antral follicle count (AFC) is a minimally invasive, easily performed test to help determine the ovarian reserve, and provides a representation of remaining follicular pool levels to assess the probability of a positive response to controlled ovarian hyperstimulation and the success of in vitro fertilization (IVF) (Maseelall, 2009). The antral follicle count (AFC) is defined as the number of follicles smaller than 10 mm in diameter detected by transvaginal ultrasound (TVS) in the early follicular phase. The AFC has been shown to be a predictor of the number of oocytes retrieved in controlled ovarian hyperstimulation protocols (Tomas et al., 1997), the cancellation rates in IVF (Frattarelli et al., 2000; Tomas et al., 1997), and for predicting pregnancy loss in IVF pregnancies (Elter et al., 2005). The AFC has also been shown to be a predictor of the number of immature oocytes retrieved for in vitro maturation (Tan et al., 2002). There is no significant difference between right-sided and leftsided antral follicle counts within the same individual (Chow et al., 2004). The AFC was shown to be a superior, or at least an equivalent, prediction tool for poor IVF response (Hendriks et al., 2007; Kwee et al., 2007) or hyperstimulation IVF response (Kwee et al., 2007) when compared to ovarian volume measurement and complex endocrine challenge tests.
A Closer Look at Indirect Measures Day 3 FSH
Day 3 FSH is believed to represent the “basal” level or non-suppressed level of FSH through ovarian feedback to the pituitary (Barnhart et al., 1998). Day 3 FSH has been credited with being a biomarker for ovarian reserve since the late 1980s, as it provided a glimpse of how well the hypothalamic-pituitary-gonadal axis is functioning (Barnhart et al., 1998). As women and their follicles age, the amount of FSH secreted increases due to the lack of responsiveness of the ovary (Perloe et al., 2000). As day 3 FSH levels climb, a diminished ovarian reserve is indicated. Fluctuation between cycles of day 3 FSH levels may be reflective of a decline in ovarian reserve (Perloe et al, 2000). Since day 3 FSH tests are an indirect test of ovarian reserve, monitoring levels may not be the best option, but this provides some insight and continues to be the most widely recognized ovarian reserve biomarker.
16
13th Annual Symposium
Focus On Quality
Thursday-Saturday, June 4-6, 2009 Rosen Center Hotel z Orlando, Florida
Session Topics z
z
z z
z
z z
z
z
z
z
z
Basic Quality Control Issues and Concerns in the Cryopreservation of Gametes and Embryos: A Look at Slow-Freeze and Vitrication Practices Mitch Schiewe, Ph.D., HCLD/ELD(ABB) Doing All the Little Things Right in the IVF Culture Environment Joseph Conaghan, Ph.D., HCLD/ELD(ABB) FDA Update New Approaches to the Management of Male Factor Etiology: To Bind or Not to Bind - Will it Impact the Success of our Patients Katie Worrilow, Ph.D. Optimizing the Quality of the IVF Laboratory Environment Jacques Cohen, Ph.D., HCLD(ABB) Oral Abstract Presentations PGD 2009: The Promise and the Problems Mark Hughes, M.D., Ph.D. Seman Analysis as it Relates to IVF: What’s Important and How do We Assure Quality? Dean Morbeck, Ph.D., HCLD(ABB) TQM for the ART Laboratory: Developing and Implementing a Program David Mortimer, Ph.D., HCLD(ABB) The Path to eSET: How do We Get There? Amy Sparks, Ph.D., HCLD(ABB) Understanding and Optimizing of Vitrication Technology: From Theory to Practical Use in the Laboratory James Stachecki, Ph.D. What is the Early Embryo Telling Us About What it Can Do? Kathleen Tucker, Ph.D.
Win a Cash Award for Your Abstract on Assisted Reproductive Technology Abstracts for both oral and poster presentation will be accepted in assisted reproductive technology and reproductive biology. There is no limit to the number of abstracts you may author. However, the rst author must be present at the poster session. Membership in the American Association of Bioanalysts and the College of Reproductive Biology is not a requirement for submitting an abstract. Oral abstract presentations will be given during the afternoon on Friday, June 5, 2009. The Conference Poster Session will be held during an evening reception on Friday, June 5. A cash award will be presented for the best assisted reproductive technology abstract presented during the session. To submit an abstract for the CRB 13th Annual Symposium, go to www.aab.org and click on 2009 AAB Annual Meeting and Educational Conference then click on “abstract submission form.” Complete the form and submit it with a copy of your abstract. Abstracts must be received no later than May 7, 2009. Abstracts received after May 7 may not be considered by the abstract review committee. Contact the CRB ofce if you have any questions.
Registration and hotel information for CRB events is available at www.aab.org, or contact the CRB by email: crb-aab@aab.org, phone 314-241-1445, fax: 314-241-1449, or by mail: CRB, 906 Olive Street, Suite 1200, St. Louis, MO 63101-1448.
The Journal of Clinical Embryology™
Volume 12, Issue 1
Testing is available on multiple automated platforms and thus is relatively fast, inexpensive, and reproducible. FSH is proven to increase with the age of follicles, and that increase is more dramatic and earlier than that of LH (Perloe et al., 2000). Historically, FSH has been the biomarker of choice; it is well studied, documented and validated which provides a level of comfort to physicians (Sharara et al., 1998). It is important to recognize some of the issues with using FSH as a biomarker for ovarian reserve testing. Between-cycle fluctuations in day 3 FSH levels make ovarian reserve estimation difficult (Perloe et al., 2000). Since lower day 3 FSH levels represent satisfactory ovarian reserve and higher levels represent declining ovarian reserve, a single day 3 FSH measurement may not be very accurate. It may be better to look at subsequent cycle day 3 FSH levels (Perloe et al., 2000). Additionally, an increased day 3 FSH level is considered a late indicator of marked decreased fertility potential (Sharara et al., 1998). It is important to continue to look for an early and more specific indicator of declining ovarian reserve and/or decreased fertility potential.
make AMH very attractive as a direct measurement of ovarian reserve (Feyereisen et al., 2006; Hehenkamp et al., 2006; La Marca et al., 2007; La Marca et al., 2006). AMH testing offers freedom to both clinicians and patients by allowing collections to be performed on any day during the menstrual cycle; a vast logistical advantage over other biomarker protocols. One recent study demonstrated not only a strong relationship between AMH and AFC, but that this relationship was stronger than the other typical biomarkers’ relationships with AFC (Feyereisen et al., 2006). In addition to being a good biomarker for follicular quantity, AMH may also be used to predict oocyte quality (Ebner et al., 2006). Women with normal reproductive performance were examined twice within an average of four years and assessed for AFC and various endocrine markers. The results demonstrated that serum AMH, followed by AFC, showed the most consistent correlation to the age-related decline of reproductive capacity (van Rooij et al., 2005). AMH has even been suggested to serve as a predictive marker for embryo implantation (Yin and Chen, 2008); however, others have not found similar results (Talebian et al., 2008). Thus, the ability to use AMH levels to predict pregnancy outcomes requires further investigation. Additional research is needed to recognize all the roles played by AMH. It will be important to investigate the following: (1) the mechanism(s) that control production of AMH within granulosa cells; (2) the role of AMH in follicle selection; (3) how AMH impacts oocyte and subsequent embryo quality for IVF; and; (4) the prospective use of AMH to predict pregnancy potential.
A Closer Look at Anti-Mullerian Hormone
As mentioned above, anti-mullerian hormone (AMH) is a member of the TGF-β superfamily and is produced by the granulosa cells of pre-antral and small antral follicles. Follicular growth is modulated by AMH, which inhibits recruitment of follicles from the primordial pool by modifying the FSH sensitivity of those follicles (La Marca et al., 2006; Visser, 2006). AMH is considered reflect the non-FSH dependant follicular growth. As a follicle matures, AMH production disappears, allowing the follicle to complete the development process during the FSH-dependant stages of growth (Visser, 2006). As a woman approaches menopause, there is a linear decline of AMH levels over time (La Marca et al., 2006; Visser, 2006). This decline is attributed to a decreasing number of follicles in the primordial pool. AMH will in all probability become the hormone of choice for assessing ovarian reserve. It has been suggested that AMH is the single best predictor of poor response for ART (Muttukrishna et al., 2005). The fact that AMH is secreted without dependence on other hormones, particularly the gonadotropins, and that AMH is expressed at a constant level independent of cycle day,
Disclosures
Beckman Coulter Inc. produces the AMH/MIS Dual Monoclonal Antibody Sandwich Enzyme Immunoassay diagnostic test system which is not currently available in the US. n
Literature Cited
Baird DT, Collins J, Egozcue J, Evers LH, Gianaroli L, Leridon H, Sunde A, Templeton A, Van Steirteghem A, Cohen J, Crosignani PG, Devroey P, Diedrich K, Fauser BC, Fraser L, Glasier L, Liebaers I, Mautone G, Penney G, Tarlatzis B. Fertility and ageing. Hum Reprod
18
What's New?
Looking for
latest
the advances?
IVF
Look no further than Mid-Atlantic Diagnostics. New STRIPPER® BP Series New Stripper BP Series with EZ-Dispenser
New UltraPure Gas Line Filter Twice the Filtering Capacity for the Same Money!
New IVF Workstations Stem Cell Workstation
The new STRIPPER BP Series and EZ-Dispense Package, the new UltraPure Gas Line Filter and the new IVF StemCell Workstation are just a few of many emerging innovations you will find at your One-Stop-Shop—Mid-Atlantic Diagnostics. As new technologies are developed and approved you'll find them right here at Mid-Atlantic. Call Mid-Atlantic Diagnostics first for all your IVF needs.
Call for more information on these and other new products! I'd like to know more about:
77 Elbo Lane • Mt Laurel, NJ 08054 Phone: 856-762-2000 • Fax: 856-762-2009 www.midatlanticdiagnostics.com
The Stripper BP Series & EZ-Dispener for Stripper Tips The UltraPure Gas Line Filter The Complete Line of IVF Workstations
Call 856-762-2000
Or fax this coupon along with your contact information to 856-762-2009
The Journal of Clinical Embryology™
Volume 12, Issue 1 Fréour T, Mirallié S, Colombel A, Bach-Ngohou K, Masson D, Barrière P. Anti-mullerian hormone: clinical relevance in assisted reproductive therapy. Ann Endocrinol (Paris). 2006;67(6):567-74. Hehenkamp WJ, Looman CW, Themmen AP, de Jong FH, Te Velde ER, Broekmans FJ. Anti-Müllerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J Clin Endocrinol Metab. 2006;91(10):4057-63. Hendriks DJ, Kwee J, Mol BW, te Velde ER, Broekmans FJ. Ultrasonography as a tool for the prediction of outcome in IVF patients: a comparative meta-analysis of ovarian volume and antral follicle count. Fertil Steril 2007;87:764-75. Kwee J, Elting ME, Schats R, McDonnell J, Lambalk CB. Ovarian volume and antral follicle count for the prediction of low and hyper responders with in vitro fertilization. Reprod Biol Endocrinol 2007;5:9. La Marca A, Stabile G, Artenisio AC, Volpe A Serum antiMullerian hormone throughout the human menstrual cycle. Hum Reprod. 2006;21(12):3103-7. La Marca A, Giulini S, Tirelli A, Bertucci E, Marsella T, Xella S, Volpe A. Anti-Müllerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum Reprod. 2007;22(3):766-71. Maseelall PB, Hernandez-Rey AE, Oh C, Maagdenberg T, McCulloh DH, McGovern PG. Antral follicle count is a significant predictor of livebirth in in vitro fertilization cycles. Fertil Steril 2009;in press. Muttukrishna S, McGarrigle H, Wakim R, Khadum I, Ranieri DM, Serhal P. Antral follicle count, anti-mullerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG. 2005;112(10):1384-90. Perloe M, Levy DP, Sills ES.Strategies for ascertaining ovarian reserve among women suspected of subfertility. Int J Fertil Womens Med. 2000;45(3):215-24. Sharara FI, McClamrock HD. The effect of aging on ovarian volume measurements in infertile women. Obstet Gynecol 1999;94:57-60. Talebian S, Licciardi F, Liu M, Grifo JA, Krey LC. Assessing anti-mullerian hormone (AMH) as a marker of ovarian response in anonymous oocyte donors: quantity or quality? Fert Steril 2008;90:S267) Tan SL, Child TJ, Gulekli B. In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular phase ultrasonography. Am J Obstet Gynecol 2002;186:684-9. Tomas CS. Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reprod 1997;12:220-3. van Rooij IA, Broekmans FJ, Scheffer GJ, Looman CW, Habbema JD, de Jong FD, Fauser BJ, Themmen AP, te Velde ER. Serum antimullerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: a longitudinal study. Fertil Steril 2005;83:979-87. Visser J. Role of anti-Müllerian hormone in follicle recruitment and maturation. J Gynecol Obstet Biol Reprod (Paris). 2006;35(5 Pt 2):2S30-2S34. Yin MN, Chen SL. AMH level in follicular fluid and serum may predict outcome of IVF-ET in PCOS patients. Fert Steril 2008;90:S377.
Update 2005;11:261-76. Barnhart K, Osheroff J. Follicle stimulating hormone as a predictor of fertility. Curr Opin Obstet Gynecol. 1998;10(3):227-32. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum. Reprod. 2007;22:1506–1512. Chow GE, Criniti AR, Soules MR. Antral follicle count and serum follicle-stimulating hormone levels to assess functional ovarian age. Obstet Gynecol 2004;104:801-4. Ebner T, Sommergruber M, Moser M, Shebl O, Schreier-Lechner E, Tews G. Basal level of anti-Müllerian hormone is associated with oocyte quality in stimulated cycles. Hum Reprod. 2006;21(8):2022-6. Elter K, Kavak ZN, Gokaslan H, Pekin T. Antral follicle assessment after down-regulation may be a useful tool for predicting pregnancy loss in in vitro fertilization pregnancies. Gynecol Endocrinol 2005;21:33-7. Faddy MJ, Gosden RG. A model confirming the decline in follicle numbers to the age of menopause in women. Hum Reprod. 1996;11:1484–1486. Feyereisen E, Méndez Lozano DH, Taieb J, Hesters L, Frydman R, Fanchin R. Anti-Müllerian hormone: clinical insights into a promising biomarker of ovarian follicular status. Reprod Biomed Online. 2006;12(6):695-703 Frattarelli JL, Lauria-Costab DF, Miller BT, Bergh PA, Scott RT. Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles. Fertil Steril 2000;74:512-7. Frattarelli JL, Levi AJ, Miller BT. A prospective novel method of determining ovarian size during in vitro fertilization cycles. J Assist Reprod Genet 2002;19:39-41.
Note to Advertisers Please request 2009 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: jceadvertising@bellsouth.net • (352) 331-5235 — The Publisher
20
THE RESULTS ARE IN.
YOU HAVE A STORAGE PROBLEM. Sooner or later, every growing IVF Center
office, and yet, assures that your patient’s
will face this problem. We’ve seen it coming for
specimens are readily available when you
years. So we’ve been refining a shipping and
need them. And best of all it costs your clinic
storage protocol to answer this rapidly
nothing. Call us today to assure your center is
growing need. It’s a turnkey solution that removes the risk and worry from your
prepared to handle the risk of potentially infectious tissue storage.
Mpls/St. Paul, Minnesota • 1-888-489-8944 | Fort Lauderdale, Florida • 1-888-953-9669 Reno, Nevada • 1-888-831-2765
www.reprot.com
© Vitrolife 1.080206.1
Everything you need for successful IVF treatment. Our product portfolio covers every step of an IVF-treatment – from the special V-Tip™ follicle aspiration needles to EmbryoGlue® for embryo transfer. We supply the instruments and media you need to give your patients the best possible care. IVF-clinics all over the world have used Vitrolife products for
more than 20 years – with great results. Our innovations include the use of a follicle aspiration needle together with an ultrasound probe, the sequential G5 Series™ and ICSI™ medium with safer recombinant albumin. Visit us at www.vitrolife.com or call 303 762 1933 to learn more.
www.vitrolife.com Vitrolife Inc., 3601 South Inca Street, Englewood, Colorado 80110, USA, Tel: +1-303-762-1933, Fax: +1-303-762-7084, E-mail: fertility.us@vitrolife.com V i t r o l i f e S w e d e n A B , F a k t o r v ä g e n 1 3 , S E - 4 3 4 3 7 K u n g s b a c k a , S w ede n . T el : + 4 6 - 31 - 7 2 1 8 0 0 0 , F ax : + 4 6 - 31 - 7 2 1 8 0 9 0 , E - m a i l : f e r t i l i t y @ v i t r o l i f e . c o m
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
Northern California Association for Reproductive Biologists (NCARB) and Bay Area Reproductive Endocrinologist Society (BARES) Joint Annual Meeting 10-9-08 Invited Speaker: Dr. Ann Kiessling
O
n September 11, 2007, California Governor Schwarzenegger approved Senate Bill No. 443 which allows for the therapeutic insemination of sperm or use of sperm in other advanced reproductive technologies in cases where the sperm donor is found reactive for HIV or HTLV-1 (http://info.sen. ca.gov/pub/07-08/bill/sen/sb_0401-450/sb_443_ bill_20070326_amended_sen_v98.pdf and http:// www.californiachronicle.com/articles/view/37530). To address how clinical policy and laboratory practice procedures can be established with assurance of staff and patient safety, NCARB and BARES devoted their joint annual meeting to this particular topic. Dr. Ann Kiessling (founder of the Bedford Stem Cell Research Foundation and faculty Harvard Medical School) was the invited speaker to address these issues to society infertility physicians and clinical embryologists. Even today, it is not well understood how HIV virus particles or retro-transcribed HIV-DNA finds its way into semen. Dr. Kiessling began her studies of HIV virus in semen and blood in the 1990s. She reported that viral burden in blood and semen is not necessarily consistent with that in blood; in another words, negative viral load in blood does not mean there is no virus in semen. Whether or not the patient has had anti-viral treatment does not predict the viral load in blood or in semen. Even under prolonged stable anti-viral treatment, semen viral load can be randomly positive or negative. Although thousands of IUI procedures have been performed in Italy and elsewhere in Europe, the follow up data is very poor. A study from France reported 4 sero-converted cases from 100 couples with timed intercourse (Natural conception in HIV-negative women with HIV-infected partners. Lanset Vol 349 March 22, 1997; Use of washed sperm for assisted reproduction in HIV-positive males without checking viral absence: A risky business? Hum Reprod, 2006, 21(2):567-8; Clinical outcome of artificial reproductive technology using
HIV-1 free sperm and the problems. 17th Congress of the Japanese Society for AIDS Research. Kobe, November 2003). Thus far, CDC has not changed its position on the use of potentially infectious semen for infertility treatment since 1991, which states that IUI using sperm from HIV infected men is not proven safe. That statement is based on women who were infected by using infectious sperm samples from sperm banks (JAMA. 1995 Mar 15;273(11):890-1). Dr. Kiessling recommended that we continue to be cautious when performing IUI even when using viral load negative semen specimens since one single infected white blood cell was observed to cause transmission in a monkey model study. Although the short term risk appears to be very low, it does not provide zero long term risk. The SPAR (Special Program of Assisted Reproduction) program at Bedford Foundation offers sperm washing and post-processing testing. Two consecutive specimens are required. Each specimen is split into two parts; half is cryopreserved and half is tested for HIV viral load. If the two specimens are positive, the patient will be referred for HIV treatment. Only negative specimens are released to the clinic for treatment, which may include IVF/ICSI or oligospermic cup insemination. IUI is not recommended due to the possibility of remaining white blood cells in the sperm sample even after washing and the potential trauma during IUI due to the insemination catheter. It is safer to leave the sperm wash sample in the cervics and allow only motile sperm to swim into the uterus. The SPAR program has been working with 28 IVF clinics providing washed sperm for fertility procedures and more than 100 babies will have been born from HIV infected men by summer of 2009. All mothers and babies are closely monitored by periodically testing. For the laboratory processing of potentially HIV infected specimens, the application of universal precautions should be sufficient since nearly everything
25
The Journal of Clinical Embryology™
Volume 12, Issue 1
can kill HIV virus. On the other hand, a vertical laminar flow (type II) hood should be used during sample processing, not the type-I horizontal hood. It is not clear whether HIV virus is present in follicular fluids. Dr. Kiessling would like to collaborate with ART laboratories in order to collect follicular fluid samples from HIV positive women. However, extra precaution should be taken when handling HEP B and HEP C positive specimens, particularly HEP C since this virus is extremely hardy and can survive in dry air for days. It must be remembered that frequently HIV positive patients are also co-infected with HEP B or HEP C or both. Public perception concerning the treatment of HIV positive couples may have a negative impact on your program. Closely following suggested guidelines of using separate incubators for HIV positive cases and only using negative tested sperm specimens is highly recommended. Nevertheless, legal consult should be sought when beginning to establish this type of 9467 Chick Embryo Ad 7/1/05 1:27 PM Page 1 program.
In the situation where the female tests positive, if the fetus is infected by HIV during gestation, it will likely result in miscarriage. The baby can be infected during the process of delivery as well.. In the US, as long as there is proactive antiviral treatment, the risk for the baby is nearly zero. Prior to the start of female patient ART treatment, it is important to make sure her viral load is non-detectable. Sperm itself has not been found to be HIV infected. However, it has not been proven that virus or retrotranscribed DNA cannot enter the egg during fertilization. Practically though, no embryo should be considered infected due to sperm interaction.
Patient SPAR logistics
Chick Embryo Extract (CEE)
Chick Embryo Extract FROZEN
Chick Embryo Extract LYOPHILIZED
Product #CE650T
Product #CE650TL
1 x 20ml
1 x 10ml
10 x 20ml
10 x 10ml
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.
Westbury, NY 1.516.333.2221 1.800.645.6264 FAX 1.516.997.4948 West Coast Office 1.800.255.9378
www.accuratechemical.com
ACCURATE CHEMICAL & SCIENTIFIC CORPORATION IT’S MORE THAN A NAME
-
IT’S A PROMISE
©2003 Accurate Chemical & Scientific Corp.
Available From Our Westbury, NY Inventory
26
Patients must first conduct a phone consult with Dr. Kiessling. Frequently this resolves any confusion or misunderstandings due to previous misinformation or instructions. It is critical that it be determined that the semen provider is serious about taking care of his health as well as partner’s health. He is questioned about medications, viral load, Hep C and any other possible co-infections. If SPAR is not convinced for any reason they are not serious about their healthcare then they are referred back to their originating physician. A semen collection kit will then be sent to the patient with instructions on how to split each ejaculated specimen, freeze, and then ship to SPAR by FedEx. If both samples test negative then both specimens will be shipped back to the clinic for treatment. If one tests positive and one negative, then another specimen will be requested and Dr. Kiessling will review the case. If both specimens test positive then the patient will be referred to HIV experts. Clinics will only receive the patient’s semen analysis and HIV test results will only be sent to patient. HIV positive men who need TESA/TESE can be tested as well. The patient can go to Boston for this procedure, or if your urologist is willing to perform the procedure, frozen TESA/TESE specimen can be shipped to SPAR for testing. SPAR is also able to test HTLV-1 in semen. Work is progressing on a HEP C virus test but it is not yet ready for clinical use. If there is extra sperm available after the treatment cycle then it can be sent back to SPAR for storage using the same shipping container. n
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
A comparison of day-3 versus day-2 and day-5 versus day-4 embryo transfers among in-vitro fertilization patients Taguchi Sagiri, M.D. Funabiki Miyako, M.D. Ujino Hiroaki, M.D. Hayashi Terumi, M.D. El Beltagy Khalid, M.D. Amano Namiko, M.T. Nakamura Yoshitaka, M.D. Corresponding author: Khalid EL BELTAGY, M.D. Oak Clinic Inc. Head Office 2-7-9, Tamade-Nishi, Nishinari-ku, Osaka, 557-0045 Japan Fax: 81-6-4398-0800 E-mail: albeltagykak3@yahoo.com Introduction
and at this stage, the embryo has compacted and is therefore developing its transporting epithelium (9). It has also activated its own genome. Several studies comparing embryo transfer on day 2 versus day 3 after oocyte retrieval but the conclusions are conflicting. One study randomized day 2 and day 3 embryo transfers based on the day of the week on which oocyte retrieval took place and found no difference in pregnancy rates (10). Another study concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3 (11) . Similarly day 4 transfers were compared to day 5 in limited studies and data outcome still unclear and needs more validation so in one study, day 4 single embryo transfers (SETs) found to be a viable option or alternative to day 5 SETs with no difference in pregnancy rates (12). As the topic of preferential transfer on day 2, 3, 4 or 5 remains controversial it was thought it would be useful to perform a retrospective study to compare implantation, pregnancy and ongoing pregnancy rates between days 2,3,4 and day5 transfers.
S
ince the start of in vitro fertilization (IVF), embryos have been transferred 2 days after IVF at the 2 to 4 cell stage. This policy was adopted because of lack of suitable culture media able to sustain embryonic development for several days, hence the uterus was supposed to provide the best environment for embryo survival. The timing of the arrival of the embryo in the uterus however is premature compared with in- vivo situations, where the embryo enters the uterus at the morula stage 4 â&#x20AC;&#x201C; 5 days after ovulation (1). Early embryonic development is controlled predominantly by factors stored in the oocyte before the initiation of embryonic gene expression during the early cleavage stages (2 â&#x20AC;&#x201C; 4). Therefore the selection process after involves delaying embryo transfer (ET) from day 2 to day 3 to observe this initial transition to embryonic gene expression control. It has been observed indeed, that large preparations of human embryos which arrest in vitro do so between the 4 - & 8-cell stage (5) In addition embryo viability can be further assessed to optimize selection by extending culture to the blastocyst stage, since only 40 % - 50 % of human embryos develop into blastocyst (6-8). One of the clear advantages of blastocyst transfer is that the embryo is returned to the uterus at a stage of development where it would normally reside (9). In vitro, the human blastocyst moves from the Fallopian Tube into the uterus on day 4 of development
Material and Methods
Patients (n=252) undergoing infertility treatment by routine IVF or intracytoplasmic sperm injection (ICSI) between July 2007 to July 2008 at Oak infertility center were included in this study.
27
The Journal of Clinical Embryology™
Volume 12, Issue 1
Ovarian stimulation protocol; Controlled ovarian stimulation was achieved by Gonadotropin releasing hormone agonist ( GnrHa ) buserlin ( Buserecur ; Fujipharma, Tokyo, Japan ) short down regulation protocol followed by recruiting the ova using pure FSH for 2 days ( Folyrmon – p; Fujipharma, Tokyo , Japan ) and human menopausal gonadotropin stimulation (HMG fujiseiyaku ; Fujipharma, Tokyo, Japan ). From the 3rd day monitoring was affected by serial ultrasound scanning and HCG ( 5,000 IU ) was administered by intramuscular injection ( HCG 5000 IU〔F〕; Fujipharma; Tokyo; Japan ) when the leading follicle reached 18mm in diameter, as described else where (30 = 11 ), oocyte retrieval was performed , 36h approximately after HCG injection through transvaginal catheter ( RWH single human OPU needle 19G / 35cm ; cook ; Queensland, Australia ) ultrasound guided route.
humidified atmosphere. For Conventional insemination, the cumulus oocyte complex (COC) were incubated with 10x10”4 motile sperm / ml of Fertilization medium (Sydney IVF Fertilization Medium, Cook; Queensland, Australia) for 17-19 h. For intracytoplasmic sperm injection (ICSI) insemination COCs were denuded in 80 IU / ml Hyaluronidase (Hyaluronidase, Sage Media ; Cooper saigical, Inc; Trambull. USA), then ICSI was performed after 16 – 18 h in Fertilization media under oil. After incubation, the next morning, the conventionally inseminated COCs were denuded and assessed, ICSI oocytes were also assessed. Both conventional and ICSI embryos with 2 pronuclei , were cultured in 30 μL drops of Cleavage medium ( Sydney IVF Cleavage Medium; Cook Medical; Queensland, Australia) under oil, till embryo transfer (ET). When embryo cleavage reached stage 7, embryos were transferred to Blastocyst Medium (Sydney IVF Blastocyst Medium; Cook Medical; Queensland, Australia) till Blastocyst transfer (BT).
Laboratory procedures; All media were purchased from cook; Queensland, Australia unless otherwise indicated. All embryo culture was performed at 37℃, 6% CO2, 5% O2 & 89 N2 in a
The First Name in
Micropipets
™
A wholly owned subsidiary of
MediCult www.medi-cult.com
www.humagenivf.com
28
The Journal of Clinical Embryology™
Volume 12, Issue 1
Embryo morphology assessment
occurring or completely compacted embryo, lacking negative morphological anomalies e.g. vacuolation, excessive fragmentation and self cavitations. Or GradeⅡ; full compacted morula with some morphological anomalies e.g. vacuolation, excessive fragmentation, self cavitation.
On day 2 and 3 embryo morphology was assessed based on the number and quality of blastomeres, the degree of fragmentation, as described by Lucinda veeck (13) (Fig.1) Day 2, Good quality embryos were determined to be either;
Moderate quality embryos; were determined to be partially compacted embryos with vacuoles or excessive fragmentation present.
Grade Ⅰ:4 - 6 cells ( blastomeres ) of equal size ; no cytoplasmic fragments or blebs Or Grade Ⅱ: 4 – 6 cells ( blastomeres ) of equal size; minor (<10%) cytoplasmic fragments or blebs Moderate quality embryos were determined to be 4-6 cells (blastomeres) of distinctly unequal size; few cytoplasmic fragments or none.
Poor quality embryos; were these embryos, with eight cells or greater, with no signs of compaction and vacuoles or excess fragments (Fig. 2). Blastocyst development was assessed on Day 5 as per the grading system described by Gardner and Schoolcraft (14). Briefly, the degree of expansion and morphology of the inner cell mass and trophectoderm were visually assessed.
Poor quality embryos, were determined to be 4-6 (blastomeres) of equal or unequal size; significant cytoplasmic fragmentation.
Day of transfer
In our clinic, based on patient counseling after discussion with her own clinician, day of transfer was decided. Basically embryo transfers were done on day 2 or 3, but in cases with history of repeated failures, ectopic pregnancy or patient’s request, day 4 or day 5 was chosen. Patients who chose to have an extended culture transfer (day 4 or day 5 ) but had only a single embryo resulting from treatment, had their transfer rescheduled to a cleavage state transfer as there was seen to be no added selection benefit of growing a single embryo to day 4 or day 5 before transfer.
Day 3, Good quality embryos were determined to be either; Grade Ⅰ: 7 – 8 cells ((blastomeres ) of equal size; no cytoplasmic fragments or blebs. Or Grade Ⅱ: 7 – 8 cells ((blastomeres ) of equal size; minor (<10%) cytoplasmic fragments or blebs Moderate quality embryos were determined to be 7-8 cells (blastomeres) of distinctly unequal size; few cytoplasmic fragments or none.
Embryo transfer
Poor quality embryos, were determined to be 7-8 (blastomeres) of equal or unequal size; significant cytoplasmic fragmentation (> 25%)
Embryo selection for transfer was based on assessment of morphology as mentioned above. In all cases under 35 years of age , a single good quality embryo ( G1 or G2 or 4AA, 4AB, ABB ) as determined by the scoring system of embryo development, dependent on the day of transfer ( Day 2 , Day 3 , Day 4 or Day 5 ) was transferred. Cases above 35 years of age, 2 good quality embryos were transferred depending on the availability and quality of embryos. In all cases, luteal – phase support was performed by the intramuscular administration of depot progesterone (Proge Depot 125 mg; Mochida co., Tokyo, Japan ) + oral
For day 4 embryos & early blaslocysts were assessed in a descriptive manner to establish the range of morphologies observed. As a result of this analysis, an in-house scoring system for day 4 embryos was developed and is described as; Good quality embryos were determined to be either Grade Ⅰ; early blastocyst, visible signs of cavitation
29
The Journal of Clinical Embryology™
Volume 12, Issue 1
micronized progesterone chlormadione acetate ( Lutoral ; Takesu co, Osaka , Japan ).
to compare qualitative variables as appropriate.
Results
A total of 252 fresh non-donor IVF cycles were queried from the database. The mean age of the patients was 35.6 ± 4.3 and the ages of the patients were similar in each transfer day group (Table 1). The patients included in each transfer group were similar with respect to the period, type of gonadotrophin stimulation protocol, number of OPU, number of ETs, indicating minimum bias in classifying patients among the various transfer groups. As for the diagnosis, number of day 1 fertilized eggs, there appeared to be significantly higher percentage of unexplained infertility among day 3 transfers when compared to day 2 ( 52.1 % versus 34.4 % ,P < 0.005 ) ( Table 1-A) , also day 1 fertilized eggs , were significantly higher among day 2 transfers when compared to day 3 ( 6.4 ± 3.5 versus 5.3 ± 2.8 , P < 0.05 ) ( Table 1-B) . Table 2, demonstrates embryological and pregnancy rates among days of transfers. Fertilization rate was significantly higher among day 2 transfers when compared to day 3 transfers (64.4 % versus 58.6 %, X2 = 6.2, P < 0.01), there were no significant difference in fertilization rate between days 3, 4 and 5 transfers. Day 4 transfers demonstrated significantly higher implantation rate when compared to either day 3 or day 5 (28.6 % versus 13 %, X2 = 4, P < 0.04, 11 %, X2
Pregnancy assessment; The serum HGC concentration was measured on day 14 after OPU by fluorescent immunoassay for the intact HCG molecule ( EV test HCG ; Nissui co. , Tokyo, Japan) at the Laboratory of our hospital. The intra-assay coefficient variation (CV) was 4.1 % for a mean HCG of 23 IU/L; 3.9% for a HCG mean of 143 IU/L, and 3.4% for a mean HCG of 707 IU/L. The inter-assay CV was 4.6% , 6.1% and 9.6 respectively, and the sensitivity was 2 IU/L, to determine the occurrence of pregnancy, as well as recording the titer on serial measures afterwards. Implantation rate; was defined as number of gestational sacs (GS) visualized on transvaginal ultrasound divided by number of embryos transferred. Initial pregnancy; was documented by rising serum HCG concentration on luteal day 14 Early pregnancy loss; was defined as pregnancy loss ＜ 12 wks Ongoing pregnancy; was defined as pregnancy ＞ 12 wks. Statistical analysis; Statistical analysis was preformed using Satisti XL 1.6 software (2006). For normally distributed quantitative variables, a t test was used to compare the means between two groups. The X2 (chi-square) test was used
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. Keep illustrations to a minimum. Embed all tables, figures, and photos into the Word documents in position appropriate to their placement. In addition, save each table, figure, or photo in individual files (Word or Excel files for tables, high resolution 300 dpi JPG files for figures and/or photos). Zip or compress all items into one file prior to submission. If email is returned due to exceeded capacity, please contact editor for alternative delivery. 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: May 1, 2009 for Vol. 12, Issue 2 (Summer 2009).
Email to: embryospeak@bellsouth.net Office Phone: (352) 331-5235 30
The Journal of Clinical Embryology™
Volume 12, Issue 1
= 4.1, P < 0.04 respectively), no significant difference in implantation rate was observed between days 2, 3 transfers (Fig 1). A trend towards higher pregnancy rate was observed among day 3 transfers when compared to days 2, 4 and 5 transfers, but the difference was not significant. The ongoing pregnancy rate was significantly higher on day 2 and 4 transfers when compared to days 3 and 5 transfers (78.1 % versus 54.1 %, X2 = 4.4, P < 0.04, 87 % versus 40 %, X2 = 4, P < 0.04 respectively) (Fig 2).
compared to the in vivo environment, even with the use of the newer sequential media that is based on the vivo environment of the embryos and for the same reason the higher pregnancy rates among day 2 embryo transfers compared to day 3 encountered by Frank et al. (16). As for the extended culture embryo transfers , it must be noted that our clinic has a single embryo transfer policy which recommends patients under the age of 35 have a single embryo transferred . However, unlike previous studies, the decision of whether a patient would have a transfer following extended culture was not based on the patient:’s response to gonadotrophins or embryo quality at cleavage stage. Rather, this decision was made clinically, prior to the commencement of the patient’s treatment. Day 4 embryo selection provided a significantly higher implantation rate compared to either day 3 or day 5 transfers in this study, in agreement with previous prospective randomized multi-center study, that found an increase in implantation rate among day 4 transfers compared with days 2 and 3 transfers (17). The transfer of day 4 embryos is beneficial in numerous ways; the embryo is returned to the uterus, to an environment where it would normally reside. This occurs post-genome activation to allow the embryo with the highest development potential to be selected from a cohort. In addition, uterine contractility is reduced at this time, all of which maximizes the potential for implantation (18,19). A demonstrated benefit of blastocyst culture and transfer is the ability to determine which embryos have not yet activated the embryonic genome, which occurs at the 4 – 8 cell stage in the human (15). Thus these embryos with limited developmental potential can be identified and excluded. This study and others (12, 21, and 22) have demonstrated that increased selection power is still evident with day 4 embryo transfer. The benefit of the use of day 4 embryo transfers is the reduction in the time that an embryo exists in vitro. It has been suggested that extending the time that an embryo is in culture may increase the susceptibility to disruption of epigenetic regulatory processes and this could explain the significant increase of ongoing pregnancy rate of day 4 transfers when compared to day 5 transfers encountered in our study. Further more, the implementation of embryo transfers on day 4 of development has given our laboratory flexibility to enable transfers to be scheduled to suit both
Discussion
Assisted reproductive technology (ART) is a numbers game with permutations that involve the transfer of multiple embryos, but the most important number in IVF is of course the number one. One embryo, one sac, one fetus and one healthy baby , in other words , the ability to choose just one embryo that will lead to successful birth of a baby is what we all crave in our profession . Seeking just this is the name of the game the “Holy Grail of IVF “(15). Our data although observational and retrospective, suggest that transfers of days 2, 4 human embryos, are viable alternatives to the embryo transfers of days 3, 5 respectively. Combining the advantage of minimal compromise of early human embryo development, as well as the selection advantage, that is well documented for extended culture were observed in this study. Our study is unique to the current literature as it is the first to stratify and compare early cleaving embryos transfer on days 2 to 3 and compacting embryos or blastocyst on days 4 to 5 outcomes in one study . Previous studies (11, 12) compared days 2, 3 or days 4, 5 transfers separately making the determination of which day to transfer would result in better outcome difficult. Since only good quality embryos based on morphology according to an international grading system were chosen, it became feasible to determine the best day for transfer at the early cleaving stages as well as the compacting and blastocyst stages. So in accordance with the Van Os HC et al. study (11) our data showed that there was no significant different in the outcome , in terms of implantation and pregnancy rates, among days 2 and 3 embryo transfers . However the ongoing pregnancy rate was higher among day 2 transfers when compared to day 3 transfers, that could be due to the exposure to the stressed environment in vitro
31
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
the clinic and the patient. Since day 2 and 3 transfers have similar pregnancy and implantation rates (but a better day 2 ongoing pregnancy rate), and since day 4 extended culture gives better embryo selection as well as better implantation and better ongoing pregnancy rates (compared to day 5 transfers), it became obvious that the ideal concept for better IVF outcome is having embryos exposed to the uterine environment for the maximum time period and in vitro environment for
minimal time period. In conclusion, day 2 (for patients with limited number of eggs) and 4 (for cases of repeated failures with many eggs who needed extended culture for better selection) embryo transfers appear to be an effective tool to maintain pregnancy rates similar to days 4 and 5 transfers while having the edge of significantly higher ongoing pregnancy rates. Besides, they offer the advantage of flexibility of transfers to be scheduled to suit both the clinic and the patient. n
Table 1: Patientsâ&#x20AC;&#x2122; demographic characteristics, significantly higher unexplained infertility cases among day 3 compared to day 2 transfers P< 0.005 (Table 1-A), day 1 fertilized eggs were significantly higher among day 2 transfers when compared to day 3 P< 0.05 (Table 1-B).
32
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
Table 2: Embryological and pregnancy rates among days of transfer, Fertilization rate was significantly higher among day 2 transfers compared to day 3 P< 0.01. Day 4 showed significant higher implantation, compared to either day 3 or day 5 (P< 0.04). Ongoing pregnancy rate was significantly higher on days 2 and 4 transfers compared to days 3 and 5 (P< 0.04).
References
Hum Reprod 2001; 16(3): 476-80. 13 ) Feil D, Henshaw RC, Lane M. Day 4 embryo Selection is equal to day 5 using a new embryo scoring system validated in single embryo transfers. Hum Reprod 2008; 23(7): 1505-1510. 14) Veeck LL. Pre-embryo Grading. In: Atlas of human oocyte and early concepts, eds. Vol. 2. Maryland: Williams and Wilkins, 1991: 121. 15) Gardner DK, Schoolcraft WB. In vitro culture of human blastocyst. In: Jansen R, Mortimer D, eds. Towards Reproductive Certainty: Fertility and Genetics Beyond. Carnforth, UK: Parthenon Publishing, 1999: 378-88. 16) Elder K, Cohen J. Assisted reproductive technology (ART) is a numbers game. In: Human preimplantation Embryo Selection, eds. preface. London: Informa Ltd 2007. 17) Frankfurter D, Keefe DL, Trimarchi JB. Day 2 embryo transfer improves IVF-ET outcome in the poor responder. Fertil Steril 2003; 80(3): s-61. 18 ) Margreiter M, Weghofer A, Kogosowski A, Mahmoud KZ, Feichtinger W. A prospective randomized multicenter study to evaluate the best day for embryo transfer; does the outcome justify prolonged embryo culture? J Assist Reprod Genet 2003; 20: 91-94. 19) Fanchin R, Righini C, Olivennes F, Taylor S, De Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13: 1968-74. 20) Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Uterine junctional zone contractions during assisted reproduction cycles. Hum Repord update 1998; 4: 440-45. 21) Braude P, Bolton V, Moore S. Human gene expression first occurs between the four and eight cell stages of preimplantation development. Nature 1988; 332: 459-61. 22) Huisman GJ, Fauser BC, Eijkemans MJ, Pieters MH. Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril 2000; 73: 117-122. 23 )Tao J, Craig R, Johnson M, Williams B, Lewis W, White J, et al. Cryopreservation of human embryos at the morula stage and outcomes after transfer. Fertil Steril 2004; 82: 108-18.
1) Harper, MJ. Gamete and zygote transport. In Knobil, E. and Neill JD, eds. The Physiology of Reproduction. VoL.1. New York: Raven Press, 1994:123-88. 2) Braude P, Bolton V, Moore S. Human gene expression first occurs between the four and eight cell stages of preimplantation development. Nature 1998; 332: 459-61. 3 ) Dobson AT, Raja R, Abeyta MJ, Taylor T, Shen S, Haqq C, et al. The unique transcriptome through day 3 of human preimplantation development. Hum Mol Genet 2004; 13:1461-70. 4) Aratley JK, Braude PR, Johnson MH. Gene activity and cleavage arrest in human pre-embryos. Hum Reprod 1992; 7: 1014-21. 5) Bolton VN., Hawes SM, Taylor CT. et al. Development of spare human preimplantation embryos in vitro: an analysis of the correlation among gross morphology, cleavage rates, and development to the blastocyst. J In Vitro Fertil Embryo Transfer 1989; 6: 30-35. 6) Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil Steril 2000; 73: 126-9. 7) Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998, 13: 3434-40. 8) Blake DA, Proctor M, Johnson NP. The merits of blastocyst versus cleavage stage embryo transfer: a Cochrane review. Hum Reprod 2004; 19:795-807. 9) Gardner DK, Lane M, Schoolcraft WB. Physiology and Culture of the human blostocyst. J Reprod Immunol 2002; 55: 85-100. 10 ) Buster JE, Bustillo M, Rodi IA, Cohen SW, Hamilton M, Simon JA, et al. Biologic and morphologic development of donated human ova recovered by non surgical uterine lavage. Am J Obstet Gynecol 1985; 153: 211-17. 11) Van Os HC, Alberda AT, Janssen-Caspers HAB, et al. The influence of the interval between in vitro fertilization and embryo transfer and some other variables on treatment outcome. Fertil Sleril 1989; 51: 360-62. 12) Laverge H, Sutter PDe, Van der ELST J, Dhont M. A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF.
33
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
Fig. 2: Scoring system developed for Day 4 embryos. Grade 1 (G1): early blastocyst, visible signs of cavitation occurring or completely compacted embryo, lacking negative morphological anomalies e.g. vacuolation, excessive fragmentation and self cavitations. Grade 2 (G2): full compacted morula with some morphological anomalies e.g. vacuolation, excessive fragmentation, self cavitation. Grade 3 (G3): partially compacted embryos with vacuoles or excessive fragmentation present. Grade 4 (G4): embryos with eight cells or greater, with no signs of compaction and vacuoles or excess fragments.
Fig. 1: Scoring system for days 2, 3 pre-embryos. (Veeck LL 1991). Grade 1 (G1): Pre-embryo with blastomeres of equal size; no cytoplasmic fragments. Grade 2 (G2): Pre-embryo with blastomeres of equal size; minor cytoplasmic fragments or blebs (< 10%). Grade 3 (G3): Pre-embryo with blastomeres of distinctly unequal size; few cytoplasmic fragments or none. Grade 4 (G4): Pre-embryo with blastomeres of equal or unequal size; significant cytoplasmic fragmentation (> 25%).
34
The Journal of Clinical Embryology™
Volume 12, Issue 1
Meetings for the Embryologist (Please send announcements for meetings you would like to see included in this listing)
Pacific Coast Symposium (PCRS) PCRS 2009 Annual Meeting “Reproductive Medicine: Innovations, Regulations, and Clinical Applications” Meeting Dates: April 22 to April 26, 2009
Hotel: Renaissance Esmeralda Resort, Indian Wells, CA Group: Pacific Coast Reproductive Society
PCRS Group Rate: $243.00 per night (single or double) plus taxes New technologies in the field of Reproductive Medicine are emerging at a rapid pace. Reproductive specialists need to understand the mechanisms of these new technologies, how they apply clinically and the potential legal and ethical issues associated with them. Regulations in the field are continually being updated and knowledge of the current regulations effecting Reproductive Medicine is essential for all practitioners. Overall Program Objectives Upon the completion of this program/course participants will be able to: 1. Interpret the significance of relevant emerging scientific medical advances in the fields of stem cells and regenerative medicine, proteomics, genomics, and metabolomics. 2. Evaluate new technologies in Reproductive Medicine including microarrays, PGD, oocyte maturation, and cryopreservation. 3. Recognize the legal and ethical ramifications of stem cell and regenerative medicine. 4. Review evolving systems for embryo culture with emphasis on moving toward single embryo transfer. 5. Appraise the scope of potential ART pregnancy outcomes. 6. Explain how defects in male meiosis might be contributing to chromosomal risks with ICSI. 7. Apply updated FDA regulations in the field of reproductive medicine. 8. Participate in open forum workshops focusing on: sperm retrieval procedures, practice management, FDA policies and record keeping, and Traditional Chinese Medicine.
35
The Journal of Clinical Embryology™
Volume 12, Issue 1
The American Society of Andrology (ASA) April 1-7, 2009 ASA 34th Annual Conference Hyatt Regency Philadelphia At Penn’s Landing 201 S. Christopher Columbus Drive Philadelphia, PA 19106 http://www.andrologysociety.com/ Program Highlights: http://www.andrologysociety.com/meetings/2009/pdf/2009%20Annual%20Meeting%20flyer.pdf Testis Workshop
Testicular Function: Levels of Regulation April 1-4, 2009 http://www.andrologysociety.com/meetings/2009/pdf/2009%20TW%20Program%20Announcement.pdf Andrology Lab April 4, 2009 Sperm Morphology: A Hands-On Workshop The Andrology Laboratories Committee will present a unique, laboratory-based training workshop with “hands-on” exploration of virtual smears for sperm morphology assessments at the 2009 http://www.andrologysociety.com/meetings/2009/pdf/ASA%20ALW%20flyer11-08.pdf ASA Special Symposium’ April 4, 2009 Issues in Urologic and Hormonal Health http://www.andrologysociety.com/meetings/2009/sym_schedule.aspx
36
The Journal of Clinical Embryology™
Volume 12, Issue 1
2nd “Meet and Greet” of the IVF Forum (the internet-based Discussion Forum Group) The weekend of April 3rd in Philadelphia. This is the weekend of the Annual American Society for Andrology Meeting. (This is unrelated to the ASA itself; just something we are doing on our own, just for fun). http://www.IVFForum.net ASA Special Symposium
April 4, 2009 Issues in Urologic and Hormonal Health http://www.andrologysociety.com/meetings/2009/sym_schedule.aspx 2nd “Meet and Greet” of the IVF Forum (the internet-based Discussion Forum Group) The weekend of April 3rd in Philadelphia. This is the weekend of the Annual American Society for Andrology Meeting. (This is unrelated to the ASA itself; just something we are doing on our own, just for fun). http://www.IVFForum.net Midwest Reproductive Symposium Chicago May 29-30 http://www.mwrs.org/ The Midwest Reproductive Symposium, entering its fifth year, has achieved recognition by faculty and experts in Reproductive Medicine as a meeting of excellence where participants can interact in an intimate setting with their colleagues. Meeting attendees have unique access to the faculty through didactic sessions, panel discussions, and workshops. Attendance doubled in 2007, reflecting the success of the program and the vision of the Chairpersons: Angeline Beltsos, MD, Barry Behr, PhD, and William Kearns, PhD. According to Dr. Beltsos, “The Symposium started in 2005 and was named for its Chicago location but open to an international audience. One drawback of major medical meetings is the limited opportunity for attendee input on topics of current debate. Our goal remains to provide a venue for the timely discussion of issues relevant to Reproductive Medicine in an interactive educational setting.” Sessions target physicians, nurses, allied health professionals, and basic scientists engaged in the practice of and research in Reproductive Medicine. Workshops were added in 2007 to give attendees access to the thought leaders in focused smaller groups.
2009 AAB Annual Meeting and Educational Conference AAB/NILA/CRB 2009 Conference In Orlando, Florida June 4-6 http://www.aab.org/conf%202007.htm AAB’s 2009 Annual Meeting and Educational Conference and the NILA and CRB Symposia will be held June 4-6, 2009, at the award-winning Rosen Centre Hotel, 9840 International Drive, Orlando, FL 32819. Room rates are $159, single or double occupancy, and $20 for an additional person. To make reservations, call the Rosen Centre toll-free at 1-800-204-7234. Be sure to mention that you are attending the AAB Conference. Reservations received after May 12, 2009, will be confirmed on a space available basis. Rooms may still be available after May 12, but not necessarily at the rates listed above. 37
The Journal of Clinical Embryologyâ&#x201E;˘
Volume 12, Issue 1
22nd Annual In Vitro Fertilization and Embryo Transfer A Comprehensive Update - 2009 Four Seasons Biltmore Santa Barbara, California July 12, 2009 to July 15, 2009 http://www.cme.ucla.edu/courses/cme-download?event%5fid=660689 This program brings together a renowned faculty to present the latest developments in every phase of in vitro fertilization and embryo management. The course addresses the needs of practicing scientists, gynecologists, reproductive endocrinologists, residents, and fellows who wish to review the specialty and update their knowledge in this rapidly changing and expanding field. The intimate setting of the conference promotes extensive contact among speakers and participants with question periods after each talk and many opportunities for informal interaction. A very extensive course syllabus developed by the course instructors will be provided and an audience response system will be used to promote interaction with the speakers. Particular attention will be devoted this year to the use of new adjuncts to gonadotropins, GnRH antagonists, endometriosis and IVF, oocyte cryopreservation, extended embryo culture, cumulus co-culture, and practical aspects of ultrasound-guided transfer technique, in vitro maturation of immature oocytes, and new microarray techniques for PGS. A mini-symposium directed by Dr. David Gardner will cover assessment of the sperm and sperm DNA integrity, non-invasive evaluation of embryo metabolism and function, embryo culture systems, and IVF laboratory regulations. Enrollment is limited. Early enrollment is strongly suggested. Fees: $1,100.00 Physicians/Laboratory Directors $825.00 Residents, Technicians, Nurses (accompanied by confirmatory letter of status from department chief or training director) Mini-symposium on the IVF Laboratory $200.00 all registrants
38
The Journal of Clinical Embryology™
Volume 12, Issue 1
42nd Annual Meeting of the Society for the Study of Reproduction “Science for the Public Good” July 18–22, 2009 David L. Lawrence Convention Center Pittsburgh, Pennsylvania
http://www.ssr.org/Meetings.shtml Accommodations: http://www.ssr.org/09Accommodations.shtml The Society is an association of scientists and physicians interested in research in reproduction. Some members are engaged in basic or applied research, while others perform clinical practice. All are dedicated to advancing knowledge of reproductive processes in animals and in humans. Members are affiliated with colleges and universities, medical and veterinary schools, medical centers, research institutes, government agencies, and industry in countries around the world. Research by SSR members focuses on important problems in human and animal reproduction, including: • • • • • • • • • •
Female and male infertility (e.g. pathophysiology, diagnosis, and treatment) Contraception Pregnancy-related disorders (e.g. pre-eclampsia, premature labor) Diseases of the reproductive tract (e.g. endometriosis, cancers of the ovary, uterus, prostate) Reproductive toxicology Lactation Animal fertility and fecundity Reproduction and conservation of endangered species Basic mechanisms controlling the function of organs involved in reproduction Mechanisms of cell differentiation and development
Laboratory Training Course: Oocyte and Preimplantation Embryo Molecular Biology Presented July 26 to August 7, 2009 at Temple University Medical School The Primate Embryo Gene Expression Resource (www.preger.org) is pleased to announce the second annual training course dedicated to advancing the study of molecular mechanisms controlling mammalian oogenesis and preimplantation embryogenesis. The goal of the two-week course is to provide hands-on training in state-of-the-art molecular techniques applicable to oocytes and early embryos of any mammalian species. As part of the overall PREGER mission of advancing non-human primate oocyte and embryo biology, we especially encourage applications from those interested in incorporating non-human primate model species into their research programs for broader basic, applied, and clinical impact. (continued next page)
39
The Journal of Clinical Embryology™
Volume 12, Issue 1
Techniques will include: mRNA isolation from oocytes and embryos, mRNA amplification, gene expression array hybridization and analysis, array data interpretation, informatics tools, & integrated pathway analysis, quantitative RT-PCR, real time PCR and real time RT-PCR, 2D PAGE and proteomics analyses, confocal microscopy and quantitative image analysis, molecular analyses of stem cells. Lectures and laboratory instruction in these methods will be given by recognized leaders in the field. Course participants will learn and apply these techniques, and may also bring their own materials for analysis to advance their individual research projects. The course will be presented July 26 to August 7, 2009 at Temple University Medical School, Philadelphia, Pennsylvania. For enquiries, pre-registration, and further information about the course please visit www.pregercourse.org. Deadline for pre-registration is June 1, 2009. For additional information or assistance, please email to klatham@temple.edu. Keith E. Latham, Ph. D., Professor The Fels Institute for Cancer Research & Molecular Biology Temple University School of Medicine 3400 N. Broad Street Philadelphia, PA 19140 tel. 215-707-7577
Featured Websites: Check out your State Law Here:
http://www.ncsl.org/programs/health/embryodisposition.htm Gamete (Egg/Sperm) and Embryo Disposition State laws on embryo disposition may address one or more of the following areas: Advanced written directives prior to the creation of frozen embryos Embryo disposition in the event of divorce or death involving a couple that has donated eggs, sperm or had embryos in vitro fertilized Options for disposition of unused embryos, including storage, disposal, donation to scientific research and adoption Human Embryos for Stem Cell research. Can you do it!
http://www.ncsl.org/programs/health/genetics/embfet.htm State laws may restrict the use of embryonic stem cells from some or all sources or specifically permit certain activities. State laws on the issue vary widely. Approaches to stem cell research policy range from statutes in California, Connecticut, Illinois, Iowa, Maryland, Massachusetts, New Jersey, and New York, which encourage embryonic stem cell research, to South Dakota’s law, which strictly forbids research on embryos regardless of the source. States that specifically permit embryonic stem cell research have established guidelines for scientists such as consent requirements and approval and review processes for projects. 40
The Journal of Clinical Embryology™
Volume 12, Issue 1
Please welcome the latest member of the JCE Editorial Board One of the UK’s leading specialists in preimplantation genetics and clinical embryology, Dr Alan Thornhill joined The London Bridge Fertility, Gynaecology and Genetics Centre as Scientific Director following periods as Director of Andrology, IVF and PGD laboratories at Mayo Clinic, USA, and Scientific Director of London Fertility Centre. Alan is a state-registered clinical scientist, HFEA recognized human embryo biopsy practitioner and board-certified high complexity laboratory director (American Board of Bioanalysis) and is Director of Bridge Genoma, the UK’s first independent laboratory dedicated to providing preimplantation genetic tests to patients and partner clinics. With over 100 published articles on fertility and genetics, his current research interests include the genetics of infertility, preimplantation genetics and reproductive pharmacogenetics. Alan Thornhill, Ph.D. HCLD Director PGD London Gynaecology & Fertility Centre Cozens House 112A Harley Street London W1G 7JH UK
athornhill@thebridgecentre.co.uk
The Journal Of Clinical Embryology™ Editorial Board Ashok Agarwal, Ph.D. HCLD Staff, Glickman Urological Institute, and Departments of Obstetrics/Gynecology, Anatomic Pathology, and Immunology Case Western Reserve University Cleveland Clinic Foundation Cleveland, Ohio 44195 Agarwaa@ccf.org David E. Battaglia, Ph.D., HCLD/ELD OHSU Fertility Consultants Oregon Health and Science University Portland, OR battagli@ohsu.edu Barry D. Bavister, Ph.D. Adjunct Professor University of Puerto Rico Medical Sciences Campus San Juan, Puerto Rico Adjunct Professor Dept. of Obstetrics & Gynecology Wayne State University, Detroit, MI, USA barrybavister@hotmail.com
Carol Brenner, PhD Departments of Obstetrics & Gynecology and Physiology Wayne State University School of Medicine Detroit MI 48201 cbrenner@med.wayne.edu
Richard G. Rawlins, Ph.D. HCLD Rush Centers for Advanced Reproductive Care Dept OB/GYN Rush Medical Center Chicago, IL 60612 Richard_G_Rawlins@rush.edu
Grace Centola, Ph.D., H.C.L.D. Professional ReproLab Consulting Macedon, New York centolag@yahoo.com
Alan Thornhill, Ph.D. HCLD Director PGD London Gynaecology & Fertility Centre Cozens House • 112A Harley Street London W1G 7JH, UK athornhill@thebridgecentre.co.uk
Kathryn J. Go, Ph.D. HCLD Reproductive Science Center One Forbes Road Lexington, MA 02421-7305 kathy.go@integramed.com David L. Hill, Ph.D. HCLD ART Reproductive Center Beverly Hills, CA embryonics@adelphia.net Thomas B. Pool, Ph.D. HCLD Fertility Center of San Antonio San Antonio, TX rpool@fertilitysa.com
41
Kenneth C. Drury, Ph.D. HCLD Dept OB/GYN University of Florida College of Medicine Gainesville, FL 32610 Editor embryospeak@bellsouth.net 352-331-5235 Fred M.W. Zander Publisher
®
FOR SUBCUTANEOUS USE ONLY BRIEF SUMMARY Please see package insert for full prescribing information. INDICATIONS AND USAGE Follistim® AQ Cartridge (follitropin beta injection) is indicated for the development of multiple follicles in ovulatory patients participating in an Assisted Reproductive Technology (ART) program. Follistim® AQ Cartridge is also indicated for the induction of ovulation and pregnancy in anovulatory infertile patients in whom the cause of infertility is functional and not due to primary ovarian failure. Selection of Patients Before treatment with Follistim® AQ Cartridge (follitropin beta injection) is initiated: 1) A thorough gynecologic and endocrinologic evaluation of the patient must be performed. The evaluation should include a hysterosalpingogram (to rule out uterine and tubal pathology) and documentation of anovulation by means of reviewing a patient’s history, performing a physical examination, determining serum hormonal levels as indicated, and optionally performing an endometrial biopsy. Patients with tubal pathology should receive Follistim ® AQ Cartridge only if enrolled in an ART program. 2) Primary ovarian failure should be excluded by the determination of circulating gonadotropin levels. 3) Careful examination should be made to rule out early pregnancy. 4) Evaluation of the partner’s fertility potential should be included in the workup procedure. CONTRAINDICATIONS Follistim® AQ Cartridge (follitropin beta injection) is contraindicated in women who exhibit: 1) Prior hypersensitivity to recombinant hFSH products 2) A high circulating FSH level indicating primary ovarian failure 3) Uncontrolled thyroid or adrenal dysfunction 4) Tumor of the ovary, breast, uterus, hypothalamus, or pituitary gland 5) Pregnancy 6) Heavy or irregular vaginal bleeding of undetermined origin 7) Ovarian cysts or enlargement not due to polycystic ovary syndrome (PCOS) 8) Hypersensitivity reactions to streptomycin or neomycin. Follistim® AQ Cartridge may contain traces of these antibiotics and may cause hypersensitivity reactions in susceptible persons. WARNINGS Follistim® AQ Cartridge (follitropin beta injection) should be used only by physicians who are experienced in infertility treatment. Changes in brand (manufacturer), type (recombinant, urinary, etc.), and/or method of administration (Follistim Pen®, conventional syringe, etc.) may result in the need to adjust the dose. Follistim ® AQ Cartridge administered with the Follistim Pen® contains a potent gonadotropic substance and delivers on average an 18% higher amount of follitropin beta as compared to lyophilized preparations administered by conventional syringe. Accordingly, a lower starting dose for gonadotropin stimulation and dose adjustments during gonadotropin stimulation should be considered for each woman treated with Follistim® AQ Cartridge (see DOSAGE AND ADMINISTRATION in the full prescribing information). Overstimulation of the Ovary During Treatment With Follistim® AQ Cartridge (follitropin beta injection) In order to minimize the hazards associated with the occasional abnormal ovarian enlargement that may occur with Follistim® AQ Cartridge therapy, the lowest effective dose should be used (see DOSAGE AND ADMINISTRATION in the full prescribing information). Use of ultrasound monitoring of ovarian response and/or measurement of serum estradiol levels can further minimize the risk of overstimulation. If the ovaries are abnormally enlarged on the last day of treatment with Follistim® AQ Cartridge, hCG should not be administered in this course of treatment, to reduce the chances of developing Ovarian Hyperstimulation Syndrome (OHSS). Ovarian Hyperstimulation Syndrome (OHSS): OHSS is a medical entity distinct from uncomplicated ovarian enlargement and may progress rapidly to become a serious medical event. OHSS is characterized by a dramatic increase in vascular permeability, which can result in a rapid accumulation of fluid in the peritoneal cavity, thorax, and potentially, the pericardium. The early warning signs of OHSS developing are severe pelvic pain, nausea, vomiting, and weight gain. The following symptoms have been reported in cases of OHSS: abdominal pain, abdominal distension, gastrointestinal symptoms including nausea, vomiting and diarrhea, severe ovarian enlargement, weight gain, dyspnea, and oliguria. Clinical evaluation may reveal hypovolemia, hemoconcentration, electrolyte imbalances, ascites, hemoperitoneum, pleural effusions, hydrothorax, acute pulmonary distress, and thromboembolic events (see WARNINGS-Pulmonary and Vascular Complications). Transient liver function test abnormalities suggestive of hepatic dysfunction, which may be accompanied by morphologic changes on liver biopsy, have been reported in association with Ovarian Hyperstimulation Syndrome (OHSS). During clinical trials with Follistim® and Follistim® AQ Cartridge therapy, OHSS occurred in 60 (5.3%) of the 1132 women treated and of these 33 (2.9%) were hospitalized. Cases of OHSS are more common, more severe, and more protracted if pregnancy occurs; therefore, patients should be followed for at least two weeks after hCG administration. Most often, OHSS occurs after treatment has been discontinued and it can develop rapidly, reaching its maximum about seven to ten days following treatment. Usually, OHSS resolves spontaneously with the onset of menses. If there is evidence that OHSS may be developing prior to hCG administration (see PRECAUTIONS-Laboratory Tests), the hCG must be withheld. If serious OHSS occurs, treatment should be stopped and the patient should be hospitalized. Treatment is primarily symptomatic and should consist of bed rest, fluid and electrolyte management, and analgesics (if needed). Hemoconcentration associated with fluid loss into the peritoneal cavity, pleural cavity, and the pericardial cavity may occur and should be thoroughly assessed in the following manner: 1) fluid intake and output; 2) weight; 3) hematocrit; 4) serum and urinary electrolytes; 5) urine specific gravity; 6) BUN and creatinine; 7) total proteins with albumin: globulin ratio; 8) coagulation studies; 9) electrocardiogram to monitor for hyperkalemia and 10) abdominal girth. These determinations should be performed daily or more often based on clinical need. OHSS increases the risk of injury to the ovary. The ascitic, pleural, and pericardial fluid should not be removed unless there is the necessity to relieve symptoms such as pulmonary distress or cardiac tamponade. Pelvic examination may cause rupture of an ovarian cyst, which may result in hemoperitoneum, and should, therefore, be avoided. If bleeding occurs and requires surgical intervention, the clinical objective should be to control the bleeding and retain as much ovarian tissue as possible. Intercourse should be prohibited in patients with significant ovarian enlargement after ovulation because of the danger of hemoperitoneum resulting from ruptured ovarian cysts. The management of OHSS may be divided into three phases: an acute, a chronic, and a resolution phase. Because the use of diuretics can accentuate the diminished intravascular volume, diuretics should be avoided except in the late phase of resolution as described below. Acute Phase: Management during the acute phase should be directed at preventing hemoconcentration due to loss of intravascular volume to the third space and minimizing the risk of thromboembolic phenomena and kidney damage. Treatment is intended to normalize electrolytes while maintaining an acceptable but somewhat reduced intravascular volume. Full correction of the intravascular volume deficit may lead to an unacceptable increase in the amount of third space fluid accumulation. Management includes administration of limited intravenous fluids, electrolytes, human serum albumin, and strict monitoring of fluid intake and output. Monitoring for the development of hyperkalemia is recommended. Chronic Phase: After stabilizing the patient during the acute phase, excessive fluid accumulation in the third space should be limited by instituting severe potassium, sodium, and fluid restriction. Resolution Phase: A fall in hematocrit and an increasing urinary output without an increased intake are observed due to the return of the third space fluid to the intravascular compartment. Peripheral and/or pulmonary edema may result if the kidneys are unable to excrete third space fluid as rapidly as it is mobilized. Diuretics may be indicated during the resolution phase, if necessary, to combat pulmonary edema. Pulmonary and Vascular Complications Serious pulmonary conditions (e.g., atelectasis, acute respiratory distress syndrome) have been reported in women treated with gonadotropins. In addition, thromboembolic events both in association with, and separate from, the Ovarian Hyperstimulation Syndrome have been reported following gonadotropin therapy. Intravascular thrombosis, which may originate in venous or arterial vessels, can result in reduced blood flow to vital organs or the extremities. Sequelae of such events have included venous thrombophlebitis, pulmonary embolism, pulmonary infarction, cerebral vascular occlusion (stroke), and arterial occlusion resulting in loss of limb. In rare cases, pulmonary complications and/or thromboembolic events have resulted in death. Multiple Births Multiple births have been reported for all FSH treatments including Follistim® (follitropin beta for injection) treatment. The patient and her partner should be advised of the potential risk of multiple births before starting treatment. PRECAUTIONS General Careful attention should be given to the diagnosis of infertility and in the selection of candidates for treatment with Follistim® AQ Cartridge (follitropin beta injection) (see INDICATIONS AND USAGE-Selection of Patients). Information for Patients Physicians must instruct patients on the correct usage and dosing of Follistim® AQ Cartridge (follitropin beta injection) in conjunction with the Follistim Pen®.
Patients should read and follow all instructions in the Follistim Pen® Instructions for Use Manual/Treatment Diary prior to administration of Follistim® AQ Cartridge. Prior to treatment with Follistim® AQ Cartridge, patients should be informed of the duration of treatment and monitoring procedures that will be required. The risks of Ovarian Hyperstimulation Syndrome and multiple births (see WARNINGS), and other possible adverse reactions (see ADVERSE REACTIONS) should be discussed. Laboratory Tests In most instances, treatment with Follistim® AQ Cartridge (follitropin beta injection) will result only in follicular growth and maturation. In order to complete the final phase of follicular maturation and to induce ovulation, hCG must be given following the administration of Follistim® AQ Cartridge or when clinical assessment of the patient indicates that sufficient follicular maturation has occurred. This may be directly estimated by sonographic visualization of the ovaries and endometrial lining and/or measuring serum estradiol levels. The combination of both ultrasonography and measurement of estradiol levels is useful for monitoring the growth and development of follicles, timing hCG administration, as well as minimizing the risk of OHSS and multiple gestations. The clinical evaluation of estrogenic activity (changes in vaginal cytology, changes in appearance and volume of cervical mucus, spinnbarkeit, and ferning of the cervical mucus) provides an indirect estimate of the estrogenic effect upon the target organs, and therefore, it should only be used adjunctively with more direct estimates of follicular development (e.g., ultrasonography and serum estradiol determinations). The clinical confirmation of ovulation is obtained by direct and indirect indices of progesterone production. The indices most generally used are as follows: a) A rise in basal body temperature b) Increase in serum progesterone c) Menstruation following the shift in basal body temperature When used in conjunction with indices of progesterone production, sonographic visualization of the ovaries will assist in determining if ovulation has occurred. Sonographic evidence of ovulation may include the following: a) Fluid in the cul-de-sac b) Follicle showing marked decrease in size c) Collapsed follicle Drug Interactions No drug-drug interaction studies have been performed. Carcinogenesis and Mutagenesis, Impairment of Fertility Long-term toxicity studies in animals have not been performed with Follistim® AQ Cartridge (follitropin beta injection) to evaluate the carcinogenic potential of the drug. Follistim® (follitropin beta for injection) was not mutagenic in the Ames test using S. typhimurium and E. coli tester strains and did not produce chromosomal aberrations in an in vitro assay using human lymphocytes. Pregnancy Pregnancy Category X: (See CONTRAINDICATIONS). Nursing Mothers It is not known whether this drug is excreted in human milk. Because many drugs are excreted in human milk and because of the potential for serious adverse reactions in the nursing infant from Follistim® AQ Cartridge (follitropin beta injection), a decision should be made whether to discontinue nursing or to discontinue the drug, taking into account the importance of the drug to the mother. Pediatric Use Safety and effectiveness in pediatric patients have not been established. Geriatric Use Clinical studies did not include subjects aged 65 and over. ADVERSE REACTIONS Assisted Reproductive Technologies (ART) Rates of adverse events from an open-label, non-controlled, multicenter study in 60 women undergoing COH for IVF or ICSI with Follistim® AQ Cartridge (follitropin beta injection) administered with the Follistim Pen® are summarized in Table 4. TABLE 4: Incidence of Adverse Clinical Experiences (≥5%) Adverse Event Abdominal pain Flatulence Abdominal pain, gynecological Nausea Breast pain, female Injection site reaction
Follistim® AQ Cartridge n=60 28% 27% 25% 17% 15% 10%
Adverse Event Abdomen enlarged Back pain Constipation Headache Ovarian pain
Follistim® AQ Cartridge n=60 8% 7% 5% 5% 5%
Ovulation Induction Rates of adverse events from an open-label, non-controlled, multicenter study in 43 clomiphene-resistant women with chronic anovulation (WHO group II) undergoing Ovulation Induction with Follistim® AQ Cartridge (follitropin beta injection) administered with the Follistim Pen® are summarized in Table 5. TABLE 5: Incidence of Adverse Clinical Experiences (≥5%) Adverse Event
Ovarian hyperstimulation syndrome Abdominal pain Injection site reaction
Follistim® AQ Cartridge n=43 9% 5% 5%
Adverse Event Sinusitis Upper respiratory tract infection
Follistim® AQ Cartridge n=43 5% 5%
The following adverse events have been reported in women treated with gonadotropins: pulmonary and vascular complications (see WARNINGS), hemoperitoneum, adnexal torsion (as a complication of ovarian enlargement), dizziness, tachycardia, dyspnea, tachypnea, febrile reactions, flu-like symptoms including fever, chills, musculoskeletal aches, joint pains, nausea, headache and malaise, breast tenderness, and dermatological symptoms (dry skin, erythema, body rash, hair loss and hives). There have been infrequent reports of ovarian neoplasms, both benign and malignant, in women who have undergone multiple drug regimens for ovulation induction; however, a causal relationship has not been established. Congenital Anomalies The incidence of congenital malformations after Assisted Reproductive Technologies (ART) may be slightly higher than after spontaneous conception. This slightly higher incidence is thought to be related to differences in parental characteristics (e.g., maternal age, sperm characteristics) and to the higher incidence of multiple gestations after ART. There are no indications that the use of gonadotropins during ART is associated with an increased risk of congenital malformations. Storage Store refrigerated, 2–8°C (36–46°F) until dispensed. Upon dispensing, the product may be stored by the patient at 2–8°C (36–46°F) until the expiration date, or at 25°C (77°F) for 3 months or until expiration date, whichever occurs first. Once the rubber inlay of the Follistim® AQ Cartridge has been pierced by a needle, the product can only be stored for a maximum of 28 days at 2–25°C (36–77°F). Protect from light. Do not freeze. For more information, call 1-866-836-5633
Follistim® and Follistim Pen® are registered trademarks of N.V. Organon
� only
Manufactured for Organon USA Inc. Roseland, NJ 07068 by Vetter Pharma-Fertigung GmbH & Co. KG Ravensburg, Germany and packaged by Organon (Ireland) Ltd., Swords Co. Dublin, Ireland ©2005 Organon USA Inc.
FOP-8009
5310202-03 3/05 24
Most Prescribed FSH in the US
a1
• Easy to use pen device2,3 • Only recombinant follicle stimulating hormone (recFSH) pen device with precise, flexible dosing in 25 IU increments4 • Available at your preferred pharmacy • Savings for self-pay patients through Design RX™. Ask your Organon representative for additional information For more information please call 1-866-836-5633 or visit www.follistim.com Please see Brief Summary on adjacent page.
INDICATIONS Follistim® AQ Cartridge (follitropin beta injection) is indicated for the development of multiple follicles in ovulatory patients participating in an Assisted Reproductive Technology (ART) program. Follistim® AQ Cartridge is also indicated for the induction of ovulation and pregnancy in anovulatory infertile patients in whom the cause of infertility is functional and not due to primary ovarian failure. IMPORTANT SAFETY INFORMATION Follistim® AQ Cartridge administered with Follistim Pen® delivers on average an 18% higher amount of follitropin beta compared to lyophilized preparation administered using conventional syringes. A lower dose should be considered when using Follistim® AQ Cartridge. Follistim® AQ Cartridge (follitropin beta injection) is contraindicated in women who exhibit: prior hypersensitivity to recombinant hFSH products, a high circulating FSH level indicating primary ovarian failure, uncontrolled thyroid or adrenal dysfunction, tumor of the ovary, breast, uterus, hypothalamus, or pituitary gland, pregnancy, heavy or irregular vaginal bleeding of undetermined origin, ovarian cysts or enlargement not due to polycystic ovary syndrome (PCOS), and hypersensitivity reactions to streptomycin or neomycin. Follistim® AQ Cartridge may contain traces of these antibiotics and may cause hypersensitivity reactions in susceptible persons. Follistim® AQ Cartridge, like all gonadotropins, is a potent substance capable of causing Ovarian Hyperstimulation Syndrome (OHSS), with or without pulmonary or vascular complications. OHSS is a medical entity distinct from uncomplicated ovarian enlargement and may progress rapidly to become a serious medical event. During clinical trials with Follistim® and Follistim® AQ Cartridge therapy, OHSS occurred in 60 (5.3%) of the 1132 women treated and of these 33 (2.9%) were hospitalized. Follistim® AQ Cartridge should be prescribed only by physicians who are experienced in infertility treatment and should advise their patients of treatment risks, including OHSS and multiple births. Multiple births have been reported for all FSH treatments including Follistim® (follitropin beta for injection) treatment. The patient and her partner should be advised of the potential risk of multiple births before starting treatment. Adverse reactions occurring in > _ 5% of patients in clinical trials undergoing ART with Follistim® AQ Cartridge included: abdominal pain 28%, flatulence 27%, abdominal pain-gynecological 25%, nausea 17%, breast pain-female 15%, injection site reaction 10%, abdomen enlarged 8%, back pain 7%, constipation 5%, headache 5% and ovulation pain 5%. Adverse reactions occurring in > _ 5% of patients in clinical trials undergoing Ovulation Induction with Follistim® AQ Cartridge included: ovarian hyperstimulation syndrome 9%, abdominal pain 5%, injection site reaction 5%, sinusitis 5%, and upper respiratory tract infection 5%. a
Based on total prescriptions from IMS data January-December 2007
References: 1. Organon USA Inc. Data on file. IMS-National Sales Perspective, January-December 2007. 2. Pang S, Kaplan B, Karande V, et al. The Follistim Pen® COH Study Group. Self-administration of Follistim® AQ (in cartridge) in women undergoing controlled ovarian hyperstimulation (COH): ease of use, safety, and efficacy of the Follistim Pen®. Reprod BioMed Online. 2003;7(3):319-326. 3. Kettel LM, Scholl G, Bonaventura L, et al. Evaluation of Follistim Pen® Injector for self-administration of Follistim® AQ (in cartridge) in women undergoing ovulation induction. Reprod BioMedOnline.2004;9(4):373-380. 4. Follistim® AQ (follitropin beta injection) Cartridge [package insert]. Roseland, NJ: Organon USA Inc; 2005.
Copyright © 2008, Schering Corporation, Kenilworth, NJ 07033.
All rights reserved.
Printed in the USA.
FQ0020
6/08
Established Efficacy Results from an open label, non-controlled, multicenter study in 60 women undergoing ovarian stimulation for IVF who had a mean duration of infertility of 4.2 years a1 • 74% of embryos obtained were grade 1 (excellent) and grade 2 (good) qualitya1 • 96% of embryos transferred were grade 1 (excellent) and grade 2 (good) qualitya1 Follistim® AQ Cartridge (n=60)
Parametersa1,2
Total mean (± SD) dose of rFSH (IU) Mean (± SD) treatment duration (days) Biochemical pregnancy rate/attempt (%) Biochemical pregnancy rate/transfer (%) Median serum estradiol level day of hCG (pg/mL) Mean (± SD) number of follicles > _ 14mm (n) Mean (± SD) number of oocytes recovered (n) Mean (± SD) number of embryos obtained Mean ± number of embryos transferred
2188.3±709.8 9.0±1.6 56.7 61.8 1423.0 range (469.5-4874.0) 10.8±5.37 13.9±10.3 7.2±5.47 2.4±0.7
a Results from an open label, non-controlled, multicenter study of 60 women undergoing controlled ovarian hyperstimulation (COH) for IVF or ICSI with Follistim® AQ Cartridge.
For more information please call 1-866-836-5633 or visit www.follistim.com Please see Brief Summary on adjacent page.
INDICATIONS Follistim® AQ Cartridge (follitropin beta injection) is indicated for the development of multiple follicles in ovulatory patients participating in an Assisted Reproductive Technology (ART) program. Follistim® AQ Cartridge is also indicated for the induction of ovulation and pregnancy in anovulatory infertile patients in whom the cause of infertility is functional and not due to primary ovarian failure. IMPORTANT SAFETY INFORMATION Follistim® AQ Cartridge administered with Follistim Pen® delivers on average an 18% higher amount of follitropin beta compared to lyophilized preparation administered using conventional syringes. A lower dose should be considered when using Follistim® AQ Cartridge. Follistim® AQ Cartridge (follitropin beta injection) is contraindicated in women who exhibit: prior hypersensitivity to recombinant hFSH products, a high circulating FSH level indicating primary ovarian failure, uncontrolled thyroid or adrenal dysfunction, tumor of the ovary, breast, uterus, hypothalamus, or pituitary gland, pregnancy, heavy or irregular vaginal bleeding of undetermined origin, ovarian cysts or enlargement not due to polycystic ovary syndrome (PCOS), and hypersensitivity reactions to streptomycin or neomycin. Follistim® AQ Cartridge may contain traces of these antibiotics and may cause hypersensitivity reactions in susceptible persons. Follistim® AQ Cartridge, like all gonadotropins, is a potent substance capable of causing Ovarian Hyperstimulation Syndrome (OHSS), with or without pulmonary or vascular complications. OHSS is a medical entity distinct from uncomplicated ovarian enlargement and may progress rapidly to become a serious medical event. During clinical trials with Follistim® and Follistim® AQ Cartridge therapy, OHSS occurred in 60 (5.3%) of the 1132 women treated and of these 33 (2.9%) were hospitalized. Follistim® AQ Cartridge should be prescribed only by physicians who are experienced in infertility treatment and should advise their patients of treatment risks, including OHSS and multiple births. Multiple births have been reported for all FSH treatments including Follistim® (follitropin beta for injection) treatment. The patient and her partner should be advised of the potential risk of multiple births before starting treatment. Adverse reactions occurring in > _ 5% of patients in clinical trials undergoing ART with Follistim® AQ Cartridge included: abdominal pain 28%, flatulence 27%, abdominal pain-gynecological 25%, nausea 17%, breast pain-female 15%, injection site reaction 10%, abdomen enlarged 8%, back pain 7%, constipation 5%, headache 5% and ovulation pain 5%. Adverse reactions occurring in > _ 5% of patients in clinical trials undergoing Ovulation Induction with Follistim® AQ Cartridge included: ovarian hyperstimulation syndrome 9%, abdominal pain 5%, injection site reaction 5%, sinusitis 5%, and upper respiratory tract infection 5%. References: 1. Pang SC, Kaplan B, Karande V, et al. Administration of recombinant human FSH (solution in cartridge) with a pen device in women undergoing ovarian stimulation. Reprod Biomed Online. 2003;7(3); 319-326. 2. Follistim® AQ Cartridge [package insert]. West Orange, NJ: Organon USA Inc.; 2005.
Copyright © 2008, Schering Corporation, Kenilworth, NJ 07033.
All rights reserved.
Printed in the USA.
FQ0020
6/08