The Journal of Clinical Embryology - Volume 12 - Issue 4 - Winter 2009 by The Journal of Clinical Embryology™

The Journal of

Clinical Embryology Volume 12, Issue 4 • Winter 2009

ISSN 1941-1901 (print) / ISSN 2152-3762 (online)

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The Journal of Clinical Embryology™

TABLE OF CONTENTS

Volume 12, Issue 4

Editor’s Corner: “EmbryoSpeak”

Editor’s Corner - “Is it Worth It?”..... 3

Is it Worth It?

Ken Drury, Ph.D., Editor

CRB Abstract: Relationship Between Seminal Plasma Heavy Metals and Sperm Function in ART...................... 5

Grace M. Centola, Ph.D.; Ian R. Hurley, Ph.D.; and Susan H. Benoff, Ph.D.

CRB Abstract: Microsecure Vitrification (µS-Vtf) of Mouse Blastocysts: Comparison to S3 Vitrification Using 0.25 Ml Straws or Cryopettes®............ 7 M.C. Schiewe, N. Nugent, K. Crawford and S. Zozula

CRB Abstract: Prognosis for Clinical Pregnancy and Delivery After Transferring Embryos Obtained from a Cohort of Incompletely Mature Oocytes at Retrieval Time................................... 9 Wiesak, T.; Grazul-Bilska, A.T.; Wikarczuk, M.; Smith, S.E.; Schinfeld, J.S.; Barmat, L.I.; Lee, A.; Somkuti, S.G.

Comparison of the Manual, IVOS, and SCA Methods for Semen Analysis Reporting..............................................11

J. Glenn Proctor, Jr., M.H.A.; William R. Boone, Ph.D.; H. Lee Higdon III, Ph.D.; William R. Boone, Ph.D.

The World’s Largest Ivf Directory....17 Zeev Schoham, M.D.

The Effect of Time in Vitrification Solution on Mouse Embryo Development, Birth Rate and Dna Damage.................................................21

Jennifer E. Graves-Herring, Ph.D.; William R. Boone, Ph.D.

Featured Website................................28 Liability Risks Associated with Mismanagement of Sperm Specimens... 29

Fethiye Sinem Karipcin, M.D.; Amjad Hossain, Ph.D.; John Y. Phelps, M.D., J.D., L.L.M.; Fethiye Sinem Karipcin, M.D.

Meetings for the Embryologist........33 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.

Ken Drury, Ph.D., Editor

e have to ask ourselves this question more and more every day. We ask it of our time; we ask it of our efforts; we ask it especially of our money. A speeding ticket on I-75 may be hard to justify, but there are many other expenditures that compete for our resources with much more efficiency and usefulness. So, this is where the Journal of Clinical Embryology gives you, once again, the opportunity to make this assessment. Since the inception of the Journal (which was previously known as the Embryologist Newsletter), ART Industry leaders committed significant funding in order to provide ART Laboratory clinical and research specific information, available free of charge, to our subscribers anywhere around the world. During that time, access to archived issues of the Journal were always available online. Even though the JCE Journal was mailed to specific subscribers, it really belonged to everyone. Today the publishing, financial, commercial landscapes have changed dramatically (thanks to Bernie Madoff and others). Many of the things people took for granted have vanished. So, what does this mean to us as individuals? As subscribers of the Journal of Clinical Embryology, it means that there will now be a subscription fee of $25.00 beginning in 2010 for our quarterly print copies and website access to the Journal ($30.00 Canada/ Mexico; $40.00 elsewhere). We trust that our past track record will lead to even better returns in the future and that your investment in the JCE “will be worth it!”. As always, I request that you bring it to my immediate attention if you feel that it is not. Please review the product presentations of our faithful advertisers in this and all Journal issues. They are the ones who truly make this valuable information available to you (please see subscription form on page 14).

In This Issue

JCE is proud to present further scientific abstracts which were presented during the 2009 AAB/CRB Annual Meeting in Orlando. Along with these abstracts you will find original data articles and informative comments designed to enhance your ART Laboratory experience. Many fine oral and poster presentations were also made available during the 65th Annual ASRM meeting in Atlanta this October. If you were one of those presenters, please consider placing your presentation into manuscript form and sending it to the Journal of Clinical Embryology. n

The Journal of Clinical Embryology™

Dear Subscribers,

Volume 12, Issue 4

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

The Journal of Clinical Embryology has been considered the primary journal covering all aspects of clinical ART laboratory interests for many years, and during this time, our subscriber base has increased considerably. However, and unfortunately, the costs of bringing you our Journal free of charge have now risen to the point that we are required to initiate a subscription fee for all our subscribers. We feel confident that our readers will want to support and maintain access to the JCE so that a minimal fee for continued service will be readily accepted. In light of the current economic realities, and projecting our growth into the future, these changes will give the JCE the opportunity to grow and expand along with the continued maturation of our clinical profession and the College of Reproductive Biology / American Association of Bioanalysts. Subscription fees will become effective January 2010 and reflect the subscriber’s rights to four issues of our hardcopy JCE as well as continued access to the JCE website, “www.embryologists.com”. The subscription fee for residents with mailing addresses within the U.S. will be $25.00; residents of Canada and Mexico $30.00 and all others $40.00. Additional details can be found on the subscription page within this issue of the Journal, as well as, on the JCE website. Very Sincerely Yours, Fred Zander, Publisher 9467 Chick Embryo Ad

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The Journal of Clinical Embryologyâ&#x201E;˘

Volume 12, Issue 4

CRB Abstracts Editorâ&#x20AC;&#x2122;s Comments: The following abstracts were presented at the Annual Meeting of the College of Reproductive Biology in Orlando, FL June 2009.

CRB Abstract: Relationship Between Seminal Plasma Heavy Metals and Sperm Function in Art Grace M. Centola, Ph.D.1; Ian R. Hurley, Ph.D.2; and Susan H. Benoff, Ph.D.2 1 LifeCell Dx, Buffalo, NY and 2Feinstein Institute for Medical Research, Manhasset, NY.

rior studies suggest that cadmium (Cd) may affect sperm fertility as a pro-oxidant leading to the generation of reactive oxygen species (ROS) and oxidative stress, while others report that infertile men have lower semen parameters and higher levels of ROS. The data below describes the competition between another pro-oxidant, inorganic lead (Pb) and zinc (Zn; an anti-oxidant) in three populations. A prospective analysis of 140 male partners of couples undergoing their first IVF cycle demonstrated an inverse relationship between seminal plasma Pb and Zn. These metals have opposing effects on sperm count, IVF fertilization rate, mannose receptor expression and mannose-induced acrosome loss. Since IVF fertilization rates following dose compensated IVF insemination are unrelated to sperm count (n = 96, r = 0.139, NS), we focused on effects on a marker of human sperm function. Mannose receptor expression is a marker for IVF fertilization rates (r = 88, n = 0.449, P<0.0001; threshold predicting reduced fertilization [<63%] = <30%), which is independent of sperm count (n = 95, r = 0.122, NS), motility (n =95, r = 0.132, NS) or normal morphology (n = 88, r = 0.082, NS). A seminal plasma Zn threshold was then determined using ROC curve analysis that best predicted poor IVF fertilization outcome (<0.515 mM).Using this threshold, we found that 2/3 of the outliers could be understood in light of the second metal. We found

that in 7/12 with normal fertilization rates and elevated lead, seminal plasma Zn levels were above the Zn ROC curve threshold. In addition, we found that in 5/6 cases of reduced fertilization and normal lead levels, seminal plasma Zn levels were well below the zinc ROC curve threshold. To determine whether Zn and lead have similar effects in other populations, we examined the ranges of these metals in seminal plasma from donors participating in an artificial insemination program (AI) and in unselected men from the general population. The data show that the ranges of Zn and Pb were similar between IVF patients, normal fertile semen donors, and unselected male population at large, while cadmium levels were significantly elevated in IVF patients (p < 0.0001). Seminal plasma Zn and Pb were inversely correlated in AI donors (n = 12; r = -0.786; p < 0.025) and also have opposing effects on AI pregnancy rates using donor sperm and on pregnancy by coitus. The IVF threshold for seminal plasma Zn (< 0.515 mM) was positively correlated with pregnancy by AI (n = 12; r = 0.648,; p< 0.03) and by coitus (n = 9; r = 0.862; p < 0.0001). Mannose receptor expression was also correlated with pregnancy following donor AI (n = 12; r = 0.765; p < 0.015) and with pregnancy by coitus (n = 13, r = 0.781, p < 0.005). These results suggest that Zn supplementation may offer an inexpensive oral therapy for the negative effects of Pb on male reproductive function. n

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The Journal of Clinical Embryology™

Volume 12, Issue 4

CRB Abstract: Microsecure Vitrification (µS-Vtf) of Mouse Blastocysts: Comparison to S3 Vitrification Using 0.25 Ml Straws or Cryopettes® M.C. Schiewe1, N. Nugent1, K. Crawford1 and S. Zozula1 1 Southern California Institute for Reproductive Sciences (SCIRS), 361 Hospital Road, Suite 433, Newport Beach, CA 92663. Introduction

Diagnostics, NJ) is a finely drawn plastic micropipette (5 cm long) connected to a bulb device, which upon sealing the tip creates a closed system. The bulb was completely depressed to load V3 solutions and the embryos before easily heat-sealing the tip prior to direct LN2 plunge. BL survival was assessed and continued BL development recorded at +24 hr.

Previously, we validated a novel VTF technique in the mouse model (Schiewe and Fahy, 2008), that we now refer to as a microSecure (µS) device. In combination with the S3 VTF system (Stachecki et al., 2008), the µS-VTF system offers sterility and security using FDA compliant devices, in addition to being safe, simple and successful in clinical application. The aim of this study was to confirm the effectiveness of the µS-VTF system compared to the S3-VTF system, and to test the technical efficacy of the new Cryopette® (CP) device.

Results

In 10 replicates of 5 BLs each/treatment, no difference was observed in recovery rates (100%) or survival rates (100%) in Expt. 1 or 2. Continued BL development in Expt. 1 decreased 10-18% overnight, but was not different (P>0.05) between the µS- and S3-VTF systems (90% and 82%, respectively).

Materials and Methods

Using frozen-thawed 1-cell mouse embryos (Embryo Tech, MN), 100 expanded to hatching blastocysts (BLs) were vitrified in each experiment using either the µS or S3-VTF system (Expt. 1) or contrasting the µS to the CP device in Expt. 2. All methods used S3 VTF solutions (V1-3: 5 min, 5min and 1 min dilutions, respectively; and T1-5: see below). The µS-VTF technique loaded BLs into shortened (cut: 2.0 cm) denuding pipettes (275-300 µm ID), detached, sterile wiped, loaded tip-end first and then LN2 plunged /stored in sealed CBS™ High Security straws. Upon thawing, internally sealed ID rods are safely read while the vitrified contents remained submerged in LN2. Once confirmed, the inner plug end was rapidly cut upon removing the straw and the VTF tip is simply poured into 15 ml of 1.0M sucrose solution (37°C; 60x15mm culture dish). Within 10-15 sec, the VTF tip contents were expelled into the T1 solution and serially pipette with the VTF tip through T5 at 5 min intervals (T1-4 at RT, T5 at 37°C) before placement in a Global® microdroplet culture system. The S3 BLs were pipette directly into labeled 0.25 ml straws, sealed and placed in a LN2 vapor tank at -100°C for 2 min before storage in LN2. For thawing, S3 straws were held at room temperature for 5 sec and submerged in a 37°C water bath for 10 sec before expelling contents into T1 solution. S3-treated BLs were then diluted and cultured as described above. The Cryopette® (CP; Mid-Atlantic

Conclusion

The µS-VTF system proved to be highly effective, similar to the established S3 VTF system. Both VTF systems offer technical simplicity aimed at reducing intra- and inter-laboratory variation, as well other quality control advantages compared to various VTF devices. The S3 system is a macro-VTF approach using 0.25 ml straws, which are standard to the IVF industry. The microSecure VTF device incorporates commonly used denuding pipettes with optimum cryosecurity. Both systems have been successfully applied in clinical IVF to definitively disprove the theory that ultrarapid cooling rates are necessary for the successful VTF of human eggs and blastocysts. The novel CP device proved to be reliable and easily applied by four different embryologists with high survival rates. The CP device requires additional viability studies and further product development to overcome QC concerns in labeling and cryostorage that ensures ease of identification and the safety of the tip, respectively.

Support

We thank T. Fortino of Mid-Atlantic Diag., Inc. for allowing us to test the CP device. n

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The Journal of Clinical Embryologyâ&#x201E;˘

Volume 12, Issue 4

CRB Abstract: Prognosis for Clinical Pregnancy and Delivery After Transferring Embryos Obtained from a Cohort of Incompletely Mature Oocytes at Retrieval Time Wiesak, T.; Grazul-Bilska, A.T*.; Wikarczuk, M.; Smith, S.E.; Schinfeld, J.S.; Barmat, L.I.; Lee, A.; Somkuti, S.G. Abington IVF and Genetics Toll Center for Reproductive Medicine, Abington, PA, USA, *Department of Animal Sciences, North Dakota State University, Fargo, ND, USA. % of mature oocytes on day of egg retrieval (32.9%, 57.5%, 69.1%, p< 0.001), in average number of 2 PNs (0.07, 1.79, 4.24, p<0.0001), in the number of cleaved embryos originated from mature oocytes on day of egg retrieval (0.036, 1.63, 3.90, p<0.001), respectively. There were no statistical differences between the three groups (LM, MX, M) in the number of oocytes that matured after overnight culture and had been inseminated on second day or in the number of 2PNs from these oocytes. However, the cleavage rate of the embryos originated from the oocytes cultured overnight in the mature group (M, 46.8%) was significantly lower (p<0.0001) then in the other two groups LM (94.2%) and MX (97.6%). Clinical pregnancy, delivery and implantation rates for group LM was 11.36%, 11.36%, 5.6%, for group MX was 13.95%, 11.63%, 4.17% and for group M was 45.24%, 33.3% and 14.61% respectively.

The objective of our retrospective study is to establish prognosis for an implantation, pregnancy and delivery rate following transfer of embryos originating either from 1) immature oocytes (GV and MI stage) with overnight in vitro maturation or 2) from mature oocytes and overnight in vitro matured oocytes that were transferred together. Total of 159 IVF cycles (1999-2008) were extracted from our database where ICSI was performed on mature oocytes at retrieval time and/or on oocytes that matured overnight. There were 24 cycles (out of 159) where embryo transfer was not performed because there were no embryos available for transfer due to lack of fertilization or cleavage. The remaining 135 cycles were grouped as follow; Group LM (n=50 late mature oocytes) - all transferred embryos originated from the oocytes that matured after overnight culture of GV and MI stage oocytes. Group MX (n=43, mixed) â&#x20AC;&#x201C; embryos from both the mature oocytes and inseminated on day of egg retrieval as well as embryos from the oocytes that matured after culturing in vitro overnight and inseminated on the second day. Group M (n=42, mature) all transferred embryos originated from retrieved mature oocytes and inseminated on the day of egg retrieval. There were statistical differences between these three groups (LM, MX and M) 1) in the average number of oocytes retrieved (6.30, 8.63, 11.62, p<0.02,), in average

Conclusion

Overnight culture of immature oocytes (GV and MI stage) resulting in next day maturity and transferable embryos might be clinically beneficial in situations where no mature oocytes are retrieved. In spite of low implantation, pregnancy and delivery rates, some patients nevertheless may have a chance for a positive outcome. Therefore, in this selected population of patients with GV and MI stage oocyte maturity at time of retrieval, overnight maturation may result in competent embryos for transfer. n

The Journal of Clinical Embryologyâ&#x201E;˘

Volume 12, Issue 4

Website: www.embryologists.com Please note that The Journal of Clinical Embryologyâ&#x201E;˘ 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.

The Journal of Clinical Embryology™

Volume 12, Issue 4

Comparison of the Manual, IVOS, and SCA Methods for Semen Analysis Reporting J. Glenn Proctor, Jr., M.H.A.; William R. Boone, Ph.D.; H. Lee Higdon III, Ph.D. Department of Obstetrics and Gynecology Greenville Hospital System University Medical Center, Greenville, South Carolina Corresponding Author: William R. Boone, Ph.D. Director of ART Laboratories • Greenville Hospital System Reproductive Endocrinology and Infertility 890 W. Faris Road, Suite 470 • Greenville, South Carolina 29605-5601 Fax: 864-455-8492 • Email: bboone@ghs.org None of the authors have any potential conflicts of interest, financial or otherwise. Poster presented at American Association of Bioanalysts, Las Vegas, Nevada, May 15-17, 2008. Manuscript submitted June 26, 2009. Accepted August 5, 2009.

semen when the sperm concentration is greater than 20 x 106. When samples fail to meet the 20 x 106 minimum, the manual method (as described by the World Health Organization (WHO) is the standard method for semen analysis (6). As technological advances are made to improve upon CASA systems, the GHSUMG andrology laboratory has begun to investigate new systems. One particular new system, the SCA (Sperm Class Analyzer; Microptic S.L., Barcelona, Spain), was compared to the validated IVOS (Hamilton Thorne Research, Beverly, MA), and the standard, non-computer assisted manual method (as described by WHO). The purpose of this study is to investigate the differences in concentration and motility among these three semen analysis methods and determine if the accuracy and efficiency is improved with the new system.

INTRODUCTION

Technological advances in computer-enhanced semen analysis reporting has given rise to a number of new systems that claim to be more accurate, efficient, and versatile in a healthcare setting, compared to older computer automated systems and manual microscopic methods. Infertility clinics and in vitro fertilization programs across the country greatly differ in their individual methods (either manually or computer assisted) for evaluating the fertility potential for male partners. This variability among methods has produced bias between different semen evaluation methods. Some studies have demonstrated a stance against replacing standard manual methods with computer assisted sperm analyzers (1,2). Proponents of computer automated semen analyzers address the ability to submit a more reproducible analysis within a very efficient system. Such a system also has the ability to generate more detailed sperm descriptions that may be indicative of fertility potential (3,4,5). The Andrology laboratory at the Hospital System University Medical Group (HSUMG) currently utilizes a computer automated semen analysis system (CASA), the Hamilton-Thorne Research Internal Visual Optical System (IVOS; version 10.8; Hamilton-Thorne Research, Beverly, MA) as its primary method for evaluating

MATERIALS AND METHODS

This prospective study was approved by the Institutional Review Committee of the Greenville Hospital System. Fifty semen specimens were collected and analyzed from patients presenting at the Hospital System University Medical Group andrology lab from December 6, 2007 to February 23, 2008. Using MicroCellTM counting chambers (Conception Technologies, San Diego, CA), measurements of sperm concentration in the range of 5.5 to 250 x 106 were performed via manual, IVOS, and SCA. These same chambers and methods were used for motility parameter comparisons. The parameter settings for the SCA were synchronized with the IVOS settings described in Johnson et al., 1996 (6). These parameter settings used were as follows: frames acquired: 7; frame

The Journal of Clinical Embryology™

Volume 12, Issue 4

rate: 30/s; minimum contrast: 8; minimum size: 6; LO/ HI size gates: 0.6 to 1.6; LO/HI intensity gates: 0.6 to 1.6; nonmotile head size: 10; nonmotile brightness (head intensity): 20; medium VAP value: 25; low VAP value: 10; slow cells motile: yes; and threshold straightness (STR): 80. Each individual analysis was performed with each method, regardless of sperm concentration or motility.

overall motility can be reported on a consistent basis between the three methods. However, the IVOS, and more predominantly the SCA, give additional sperm parameters that are used in making decisions regarding a patient’s treatment protocol. The SCA also has advanced capabilities that allow for digital recording of semen samples and compatibility to modern electronic medical records (The newest version 12 of the Hamilton-Thorne IVOS also has these capabilities; the IVOS used in this study has been in operation for 14 years in our laboratory and is out dated from these technological standpoints). The results from this study indicated that while the concentration and overall motility parameters were not significantly different between the three analyses methods, the forward progression parameter did differ between the methods. This difference is potentially related to the fact that the three methods all analyze sperm characteristics by different mechanisms. The standard manual method assigns forward progression values from a subjective point of view that is independent between technicians. The values assigned are a result of an individual’s or a team of technician’s definition of sperm velocity. This method of analysis is different from the way that a CASA system operates, where computer software is actually measuring different angles of sperm velocity. However, the SCA and the (continued on page 15)

RESULTS

Data are summarized in Table 1. The data did not differ among the manual, IVOS, and SCA in sperm concentration (P=0.9). While the same holds true for overall motility reporting with the manual, IVOS, and SCA (P=0.9), data did differ among the three semen analysis methods in the motility parameter of forward progression at the rapid (4), medium (3), and slow (2) levels (P=.0001 at each level). However, data did not differ among the three analysis methods at the forward progression static level, which classifies non-motile sperm (P = 0.4).

DISCUSSION

Technological advances in CASA systems have been deemed clinically valuable due to the ability of generating detailed descriptions of sperm capacity that are not achieved with the standard manual count. As our study indicates, sperm concentration and

Table 1 Sperm parameter comparisons for manual, IVOS, and SCA semen analysis methods. Parameter

Manual

IVOS

SCA

P-Valuea

Concentration (million/mL; mean ± SD)

72.9 ± 53

77.6 ± 54

76.8 ± 52

0.9

Motility (%; mean ± SD)

56 ± 10

57 ± 14

56 ± 15

0.8

Rapid Progression (%; mean ± SD)

28 ± 15

39 ± 10

35 ± 12

<0.001

Moderate Progression (%; mean ± SD)

23 ± 8

16 ± 6

14 ± 4

<0.001

Slow Progression (%; mean ± SD)

7±5

2±1

7±2

<0.001

41 ± 12

43 ± 14

44 ± 15

0.4

Static (%; mean ± SD)

These continuous data were analyzed with Student’s t-test.

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(continued from page 12)

Your clients expect flawless service. So do ours.

IVOS differ from the way that the optics is used to decipher this information. The SCA software uses positive phase objectives. These objectives produce an image with dark sperm tails and bright sperm heads that are characterized by a halo effect that defines individual sperm. The IVOS system utilizes negative phase objectives that produce a contrast of bright sperm against a dark field. These two systems have been deemed as effective CASA systems, the debate of which objective configurations are the optimum methods for semen analysis is under review.

CONCLUSIONS

The manual, IVOS, and SCA methods for reporting semen analysis results produced comparable results regardless of different degrees of sperm concentrations. Overall motility values also were compared among the methods tested. However, there were significant differences in the motility aspects of the forward progression parameter. These differences in forward progression may be explained by the different operational variables associated with each individual method. n

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ACKNOWLEDGEMENTS

Tom Kenny from Hamilton Thorne Research and CW Sturgeon from Fertility Technology Resources, Inc. that distributes the SCA on behalf of Microptic S.L. are thanked for their technical and editorial assistance.

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REFERENCES

Neuwinger J, Knuth UA, Nieschiag E. Evaluation of the HamiltonThorn 2030 motility analyzer for routine semen analysis in an infertility clinic. Int J Androl 1990;13(2):100-9. Sukcharoen N, Aribarg A. Evaluation of the Hamilton-Thorn motility analyzer for routine semen analysis. J Med Assoc Thai 1995:78(4):182-90. Centola GM. Comparison of manual microscopic and computerassisted methods for analysis of sperm count and motility. Arch Androl 1996;36(1):1-7. Barratt CL, Tomlinson MJ, Cooke ID. Prognostic significance of computerized motility analysis for in vivo fertility. Fertil Steril 1993;60(3):520-5. Joshi N, Kodwany G, Balaiah D, Parikh M, Parikh F. The importance of computer-assisted semen analysis and sperm function testing in an IVF program. Int J Fertil Menopausal Stud 1996;41(1):46-52. Johnson JE, Boone WR, Blackhurst DW. Manual versus computer-automated semen analyses. Part I. Comparison of counting chambers. Fertil Steril 1996;65(1):150-5.

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The Journal of Clinical Embryology™

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The World’s Largest Ivf Directory Zeev Schoham, M.D. Professor, Department of Obstetrics and Gynecology, Kaplan Medical Center, Rehovot, Israel. [zeev@cc.huji.ac.il] WWW.IVF-WORLDWIDE.COM IVF-Worldwide.com was created as a partnership, formed between Zeev Shoham and Milton Leong, as a contribution to the medical community. The website was launched on April 2010, and currently the global directory already comprises over 2940 units! Directory participation is free of charge, and will remain so in the future. IVF-Worldwide.com website offers a platform for a virtual IVF community. The vision for www. IVF-Worldwide.com is to give to its members free and complete information about any IVF unit in the world and to provide a suitable platform for dialogue and advanced research on IVF issues between IVF medical professionals. IVF-Worldwide.com mission is to be the largest and most comprehensive IVF-focused website for doctors, embryologists, nurses and social workers. To connect doctors and specialists from IVF centers worldwide in order to encourage dialogue, discuss special treatments and medicine and advance research on IVF issues The Educational Center The Educational Center available on the website will be a forum where experts in the field of IVF provide commentary and information so as to serve as a global reference for different aspects of the IVF process. Information will be available as presentations, movies and medical publications. Additionally a professional forum will allow communication between IVF professionals. Check out the Educational Center at http://www.ivf-worldwide.com/Education/educationcenter.html. Another feature of the Educational Center is comprehensive information on drugs used in the fertility treatment process. You are welcome to create a link from your website to the Educational Center for the benefit of your patients at http://www.ivf-worldwide. com/Education/guideline-for-drug-administration. html. For your convenience, you may use the attached logo in your website in order to provide the link to this

Educational Center information. IVF Worldwide Survey – new service for the IVF treating physicians A monthly survey on treatment modalities will be launched on the website and results will be published on a monthly basis. Results from the survey will provide a global perspective on treatment protocols and drugs used in the process. The first survey will focus on Luteal Phase Support. No center-specific information will be published rather the information will be consolidated into a general report of treatment approaches and protocols and posted on the website. n

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The Effect of Time in Vitrification Solution on Mouse Embryo Development, Birth Rate and Dna Damage Jennifer E. Graves-Herring, Ph.D. ETSU Fertility Services • 325 North State of Franklin Road First Floor, Department of OBGYN • Johnson City, TN 37604 jgraves@clemson.edu

William R. Boone, Ph.D. Greenville Hospital System, University Medical Group Department of OBGYN, Division of REI • 890 West Faris Road, Suite 470 Greenville, SC 29605 bboone@ghs.org

Manuscript Submitted: July 7, 2009 Revised Accepted: October 24, 2009

et al., 2002; Kuwayama et al., 2005). This can be difficult, especially since some vitrification devices have an extensive learning curve before the technique is mastered. Even with practice, one may not be able to quickly pick up embryos and vitrify within the time required; therefore, determining the length of time that embryos can safely reside in vitrification solution is important. One way to determine the toxicity of a vitrification solution is to analyze embryos using the Comet Assay to detect DNA damage following exposure to these high osmotic solutions. This assay was first performed by Ostling and Johanson (1984) to observe murine lymphoma cells and later modified by Singh et al. (1988) to observe “single-stranded DNA breaks and alkali-labile sites.” The premise for the Comet Assay is that damaged DNA strands will migrate out of a cell during electrophoresis to create the tail segment of the comet (the longer the tail, the more damage present); whereas, undamaged DNA will remain in the cell creating the head of the comet. This assay has been used to detect DNA damage in bovine oocytes (Chung et al., 2007) as well as hamster (Takahashi et al., 1999), bovine (Takahashi et al., 2000) and mouse (Fabian et al., 2003) embryos. Stowinska et al. (2008) and Kalthur et al. (2008) used the Comet Assay to analyze DNA damage in cryopreserved sperm. To our knowledge, our study is the first to use the Comet Assay to detect DNA damage in cryopreserved mouse embryos, although other researchers (Sohn et al., 2002; Ramezani et al., 2005; Kader et al., 2009) have used the terminal

Introduction

Vitrification is a process by which cells can be frozen in such a way that a glass-like or vitrified state is obtained. This process eliminates the formation of intracellular ice crystals which can damage organelles within the cell and cell membranes. Vitrification of mouse embryos was first reported in 1985 (Rall and Fahy). Since then, this technique has been extended to early-cell embryos (two- to eight-cells) in cattle (Vajta et al., 1997) and humans (Mukaida et al., 1998) as well as blastocysts for cattle (Park et al., 1999), mice (Lane et al., 1999), humans (Yokota et al., 2001), monkeys (Yeoman et al., 2001) and pigs (Misumi et al., 2003). One concern regarding vitrification is the exposure of the embryos to high-osmolarity cryoprotectants because of the detrimental effects that have been observed. Rall (1987) determined that eight-cell mouse embryos remaining in vitrification solution containing dimethyl sulfoxide, acetamide and propylene glycol at 4°C for 10 to 15 minutes were able to survive, but none were able to survive after remaining in the same solution for 30 minutes. Exposure of Day-4 mouse embryos to glycerol, dimethyl sulfoxide or propylene glycol for 20 minutes, was shown to be toxic (Ali and Shelton, 2007). To avoid embryo exposure to high concentrations of toxic cryoprotectants for extended periods, vitrification protocols generally require that embryos remain in the vitrification solution for only 1 to 2 minutes (Lieberman

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deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) Assay to detect DNA damage in mouse embryos. The objectives of this study are 1) to determine the length of time in vitrification solution needed to observe a reduction in blastocyst rate; 2) to determine the effect of time of exposure in vitrification solution on blastocyst rate and birth rate; and 3) to determine the blastocyst rate and percentage of embryos demonstrating DNA damage due to extended exposure in vitrification solution.

specified amount of time, the embryos were collected and moved through a series of four thawing solutions at 37°C. The first solution consisted of DPBS with 1 M sucrose (Sigma) and 20% Synthetic Serum Substitute (SSS; Irvine Scientific, Santa Ana, CA). The second solution consisted of DPBS with 0.5 M sucrose and 10% SSS. The third solution consisted of DPBS with 0.25 M sucrose and 5% SSS and the fourth solution consisted of DPBS with 0.125 M sucrose and 2.5% SSS. The amount of time that the embryos remained in each solution was 2 minutes, 3 minutes, 5 minutes and 5 minutes, respectively. Embryos then were placed into 50 µL drops of Human Tubal Fluid (HTF; Irvine Scientific) overlaid with washed mineral oil and cultured in an incubator at 36.7°C with 5% CO2 and air. After 72 hours, blastocyst rates were determined.

Materials and Methods

Male and female mice (B6C3F1) were purchased from Jackson Laboratories (Bar Harbor, ME). This strain of mice was chosen because it has demonstrated good survival results when the embryos were slow-cooled and thawed. All mice were handled according to an Institutional Animal Care and Use Committee protocol for this project. Once acclimated, the female mice were injected with pregnant mare serum gonadotrophin on Day 1, and then injected with human chorionic gonadotrophin and mated on Day 3. On Day 5, the female mice were euthanized, the oviducts removed and embryos were retrieved at the two-cell stage. The same procedure to collect two-cell mouse embryos was used in each of the three experiments.

Experiment 2: Determine the effect of vitrification solution on blastocyst rate and birth rate when embryos remain in vitrification solution for 32 minutes. This experiment used the same media and methods as described for Experiment 1. The difference was that all embryos remained in vitrification solution for 32 minutes prior to their exposure to the thawing solutions. Also, this experiment used control embryos which were collected and placed into HTF without being exposed to any vitrification or thawing solutions. After culturing for 72 hours, the blastocyst rate was determined for the controls and 32-minute exposure embryos. Cultured embryos were divided into three groups. The first group was control blastocysts. The second group consisted of early and expanded blastocysts from the 32-minute exposure group, and the third group consisted of four-cell to morula-stage embryos from the 32-minute exposure group. Embryos from all three groups were transferred into psuedopregnant synchronized recipient mice.

Experiment 1: Determine the length of time in vitrification solution needed to observe a reduction in blastocyst rate. The first experiment consisted of three trials. Trial 1 had two-cell mouse embryos remaining in vitrification solution for 1, 2, 4 or 8 minutes. Trial 2 had embryos remaining in vitrification solution for 1, 2, 4, 8, 16 or 32 minutes. Trial 3 had embryos remaining in vitrification solution for 1 or 32 minutes. For all trials, two-cell mouse embryos were exposed to a medium consisting of Dulbecco’s Phosphate Buffered Saline without calcium and magnesium (DPBS; In Vitro Care, San Diego, CA), 7.5% ethylene glycol (Sigma, St. Louis, MO) and 7.5% dimethyl sulfoxide (DMSO; Sigma) for 3.5 minutes. The embryos then were transferred to a vitrification solution consisting of DPBS with 15% ethylene glycol and 15% DMSO (Graves-Herring and Boone, 2009). Both media were at 4°C immediately prior to use and remained at room temperature thereafter. After remaining in vitrification solution for the

Experiment 3: Determine the blastocyst rate and percentage of embryos demonstrating DNA damage due to exposure to vitrification solution for 32 minutes followed by vitrification and warming. Similar to Experiment 1, embryos were exposed to the vitrification solution; however, for this experiment they remained in vitrification solution for either 1 minute or 32 minutes. The embryos then were placed 10 at a time into 150 µm Stripper Tips® (MidAtlantic

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Diagnostics Inc., Marlton, NJ), which were sealed at both ends using a Cryo Bio Systems SYMS Sealing System (Cryo Bio Systems, L’Aigle, France). Within 1 minute of transferring the embryos into the 15% ethylene glycol and 15% DMSO vitrification solution, the sealed Stripper Tips® were placed vertically into a goblet on an aluminum cane, which resided in 4 liters of liquid nitrogen in a Styrofoam container. A second goblet was inverted and attached to the top of the cane to secure the Stripper Tips® within the goblet. The canes were covered with cardboard sleeves and transferred to a storage tank containing liquid nitrogen. To thaw the vitrified embryos, Stripper Tips® were removed from liquid nitrogen and exposed to air. The area within the Stripper Tip® containing the embryos was thawed quickly by rubbing this location between the thumb and index finger for 2 to 3 seconds. Both ends of the Stripper Tip® were cut off and the embryos were expelled from the device with the aid of a 0.1 mL bolus of media using a 25 gauge needle attached to a 1 mL syringe. The embryos were collected and moved through the same series of four thawing solutions at 37°C described for Experiment 1. Once thawed, embryos from the 1-minute and 32-minute exposure groups were placed into designated 50 µL drops of Human Tubal Fluid (HTF; Irvine Scientific, Santa Ana, CA) overlaid with washed mineral oil and cultured in an incubator at 36.7°C with 5% CO2 and air. On the same days that the embryos were thawed, fresh two-cell mouse embryos were collected for controls (cultured in HTF) and for positive controls (cultured in HTF with 1% hydrogen peroxide to induce DNA damage per instructions using Comet Assay Kit). Controls and positive controls embryos were cultured in the same environment as described above. After 72 hours, blastocyst rates for controls, positive controls, 1-minute and 32-minute exposure groups were determined. Chi-square analyses were performed on the blastocyst rate for each group. Once blastocyst rates were determined, the Comet Assay Kit (Trevigen®, Gaithersburg, MD) was used to evaluate the four groups of embryos at the blastocyst stage (controls, positive controls, 1-minute exposure and 32-minute exposure) for the presence of comet tails. Manufacturer’s recommendations to perform the assay were optimized for our laboratory. Embryos were placed into 75 µL of melted agarose. The melted agarose containing the embryos then was placed on a

Comet Assay slide. The slide was held at 4°C in the dark for 30 minutes and then placed into a 4°C lysis solution for 1 hour. After this incubation, the slide was placed into an alkaline solution at 4°C for 30 minutes. This was followed by two 5 minute rinses of the slide with Tris-Borate-EDTA (Fisher Scientific, Pittsburg, PA). Electrophoresis was performed for 20 minutes at 20 volts and 300 amps. The slide was rinsed in alcohol for 5 minutes, allowed to dry and then placed into a desiccator. At the time of analysis, 50 µL of SYBR® Green I was added to the slide and the slide was then observed using fluorescence microscopy with a fluorescein isothiocyanate filter. Each embryo was observed and an image was captured using Slide Book Software (Intelligent Imaging Innovation, Inc., Denver, Colorado). All embryo images were printed and examined for the presence or absence of a comet tail. Chi-square analyses were performed on the percentage of observed comet tails for all four embryo groups.

Results

Experiment 1: The first trial produced a 100% (10/10) blastocyst rate for each of the 1-, 2- and 8-minute exposure time (in vitrification solution) groups. The 4-minute exposure time group produced a 78% (7/9) blastocyst rate. The second trial produced a 100% blastocyst rate for the 1 (n=9), 2 (n=8), 4 (n=10) and 8 (n=10) minute exposure time groups. The 16-minute

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exposure time group produced a 95% (19/20) blastocyst rate and the 32-minute exposure time group produced a 44% (8/18) blastocyst rate. The third trial produced 100% (22/22) blastocyst rate for the 1-minute exposure time group and a 72% (38/53) blastocyst rate for the 32-minute exposure time group (Table 1). Experiment 2: The blastocyst rate was significantly different (P < 0.001) between the control embryos (no exposure) and the 32-minute exposure time embryos (Table 2). After the embryos were split into three groups (control, 32-minute blastocyst stage and 32-minute multi-cell and morula stage) and transferred to recipient females, there was no significant difference (P = 0.3) in birth rates between the control embryos (no exposure) and the second group of embryos, which included early and expanded blastocysts (Table 3). However, there was a significant difference (P < 0.05) in birth rates between the second group and third group of embryos, which included four-cell to morula group. There was also a significant difference (P < 0.05) in birth rates between the control embryos and the third group of embryos. Experiment 3: There was no significant difference (P = 0.316) between the blastocyst rates for the 1-minute exposure group and 32-minute exposure group of vitrified and thawed embryos (Table 4). In contrast, the control group did demonstrate a difference in blastocyst rates (P < 0.001). In addition, the percentage of

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embryos that presented comet tails for the controls, positive controls, 1-minute exposure and 32-minute exposure group are shown in Table 4. The 1-minute exposure group was not significantly different than the control group (P = 0.174) but was significantly different from the 32-minute exposure group (P < 0.001) and the positive controls (P < 0.001). The 32-minute exposure group was

significantly different from the control group (P < 0.001) and the positive control group (P = 0.016).

Discussion

This research explored the effect of time of two-cell mouse embryos in vitrification solution. There is a plethora of published research of vitrification that described various devices,

Table 1. Time in Vitrification Solution (Experiment 1).

Table 2. Time in Vitrification Solution Blastocyst Rate (Experiment 2).

a,b

Different superscripts indicate statistical difference (P < 0.05).

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protocols and recipes. There are also various species that have been used as models and the mouse model also includes many strains. We have chosen the vitrification method and solutions that are familiar to our facility (Graves-Herring and Boone 2009) and have chosen the mouse strain that we use to perform toxicity testing. Although we will be comparing our research to others, their solutions, methods, media temperatures, cell-stage and strains of mice may not be the same; therefore, the conditions are not equivalent.

Table 3. Time in Vitrification Solution Birth Rate (Experiment 2).

Experiment 1 explored the amount of time (1, 2, 4, 8, 16 or 32 minutes) an embryo could be exposed to vitrification solution before a reduction in blastocyst rate was observed. This experiment was necessary to determine the exposure time for Experiment 2 and 3. For this preliminary study, the blastocyst rate was considered reduced if it was less than or equal to 80%. (A blastocyst rate of 80% is considered normal development for two-cell mouse embryos cultured in our laboratory.) In this experiment embryos were not vitrified, but were instead exposed to vitrification and thawing solutions and then cultured to the blastocyst stage. In Trial 1 a reduced blastocyst rate was observed for the 4-minute exposure group (78%) but not for the 8-minute exposure group (100%). In Trial 2, a slight decrease in blastocyst rate was observed for the 16-minute exposure group (95%), but only the 32-minute exposure group demonstrated the defined reduction in blastocyst rate (44%). In Trial 3, a decrease was observed at the 32-minute exposure group (72%). The reduction in blastocyst rate observed at 32 minutes determined the extended time in vitrification solution for Experiment 2 and 3. Rall (1987) determined that eight-cell mouse embryos remaining in vitrification solution containing dimethyl sulfoxide, acetamide and propylene glycol at 4Â°C for 10 to 15 minutes were able to survive, but none were able to survive after remaining in the same solution for 30 minutes. Ali and Shelton (2007) demonstrated exposure of Day-4 mouse embryos to glycerol, dimethyl sulfoxide or propylene glycol for 20 minutes to be toxic (blastocyst rate of 27.3% [12/44]). In our study the 16-minute exposure group was able to produce a 95% blastocyst rate. Although we did not observe blastocyst rates for times between 17 to 20 minutes, we demonstrated that 44% (Trial 2) and 72%

Different superscripts indicate statistical difference (P < 0.05). a,b

Table 4. Time in Vitrification Solution Blastocyst Rate and Blastocysts Demonstrating Comet Tails (Experiment 3).

Different superscripts in groupings indicate statistical difference (P < 0.05).

a,b,c

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Experiment 1 the blastocyst rate for 32-minute exposure embryos was 44% (Trial 2) and 72% (Trial 3), we had a higher blastocyst rate in Experiment 2 (76%). The difference in blastocyst rates could be the result of the low number of embryos (n=18 [Trial 2]; n=53 [Trial 3]) used in Experiment 1. In Experiment 2, the number of embryos used in the 32-minute exposure group was four times the amount of embryos used in the control group (no exposure; 255 vs. 64). In order to transfer 16 embryos to each designated recipient mouse (8 to each oviduct), we needed to ensure there would be adequate numbers of embryos available for transfers of the second group (32-minute blastocyst group), and third group (32-minute multi-cell to morula stage group). There was no significant differences (P = 0.3) between the birth rates for the controls (31% [15/48]) and the second group (42% [20/48]), indicating that those embryos that remained in vitrification solution for 32 minutes and produced blastocysts could produce pups at the same rate as embryos that were not exposed to vitrification solution. In contrast, those embryos that remained in vitrification solution for 32 minutes but were unable to produce blastocysts (32-minute multi-cell to morula stage group) were unable to produce pups (2% [1/48]) similar to the controls (no exposure; 31% [15/48]; P < 0.05). Experiment 3 demonstrated that although there was no difference in blastocyst rates between the 1-minute exposure group and 32-minute exposure group in blastocyst rate, the 32-minute exposure group embryos had significantly more DNA damage as determined by the Comet Assay. A pitfall of the Comet Assay is its inability to specify the exact type of DNA damage. If there is damage to individual blastomeres, degeneration of blastomeres, or degeneration of polar bodies, the Comet Assay cannot delineate these differences. Even though the Comet Assay does not pin-point the origin of the DNA damage, it can provide useful information as to the extent of the damage present by the length of the comet tail. A major concern with vitrification has been that the solutions are toxic. Those that are performing the procedure must be capable of quickly moving embryos through vitrification solution and loading/ placing them into/on a device for storage, sealing the device (if applicable) then quickly plunging the device into liquid nitrogen. In some cases, it is recommended

Figure 1. Mouse blastocyst with some DNA damage (as indicated by the small comet tail).

Figure 2. Mouse blastocyst with a large amount of DNA damage (as indicated by the large comet tail).

(Trial 3) of embryos exposed to vitrification solution for 32 minutes were able to develop. This is a higher developmental rate than either Rall (1987) and Ali and Shelton (2007) observed, indicating that the vitrification solutions in this study may not be as toxic. Experiment 2 demonstrated that the blastocyst rate for the 32-minute exposure group was 76% (193/255) which was significantly different (P < 0.001) than the control group (no exposure; 95% [61/64]) indicating that the extended time in vitrification solution inhibited blastocyst growth. Although we determined in

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that this process be completed in 90 to 110 seconds. The vitrification solution used in this study has shown that mouse embryos can remain in the solution for 32 minutes and the embryos have the capacity to produce blastocysts (although at a decreased rate). This gives some leeway to those individuals that are learning the procedure and may not be able to perform the vitrification process within a limited amount of time. This study is just one of many that are needed to evaluate the exposure time to vitrification solutions. Some future studies should include a more extensive study into exposure time. We only ran three trials to determine the 32 minute exposure time. More trials with times within the 16 to32 minutes should be performed. For Experiment 2 we observed embryos that were not exposed to vitrification solution to those that were exposed for 32 minutes. Another future study should include exposing embryos to vitrification solution for 1 minute and comparing the blastocyst and live birth results to those exposed for 32 minutes and those not exposed. Not only should the experiment include the process of exposing the embryos to solution, but should also include the vitrifying process and results of not vitrifying to vitrifying should be compared. This would help explain why, in Experiment 2, we observed a 76% blastocyst rate and in Experiment 3 we observed a 51% blastocyst rate for the 32-minute exposure groups. With this future study, we might be able to conclude if this was due to the vitrifying process. If the Comet Assay also as used, we potentially could determine if the vitrifying process causes more DNA damage than just exposure to solutions. In summary, exposure of mouse embryos to vitrification solution for 32 minutes does cause a decrease in blastocyst rate compared to embryos that are not exposed to vitrification solution. However, if the embryos grow to the early or expanded blastocyst stage and are transferred into recipients, these embryos can produce pups at the same rate as those that are not exposed to vitrification solution despite the higher rate of DNA damage demonstrated by the 32-minute exposed embryos. n

Fabian D, Rehak P, Czikkova S, Il’kova G, Baran V, Koppel J. Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay. Theriogenology. 2003;60:691-706. Graves-Herring J, Boone WR. Blastocyst rates and live births from vitrification and slow-cooled two-cell mouse embryos. Fertil Steril. 2009;91:920-4. Kader A, Agarwal A, Abdelrazik H, Sharma RK, Ahmady A, Falcone T. Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing. Fertil Steril. 2009;91(Suppl 5):2087-94. Kalthur G, Adiga SK, Upadhya D, Rao S, Kumar P. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia. Fertil Steril. 2008;89:1723-7. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. RBM Online. 2005;11:608-14. Lane M, Schoolcraft WB, Gardner DK, Phil D. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril. 1999;72:1073-8. Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipet. RBM Online. 2002;4:146-50. Misumi K, Suzuki M, Sato S, Saito N. Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method. Theriogenology. 2003;60:253-60. Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod. 1998;13:2874-9. Ostling O, Johanson KJ. Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem and Biophys Res Commun. 1984;123:291-8. Park SP, Kim EU, Kim DI, Park NH, Won YS, Yoon Sh, Chung KS, Lim JH. Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids. Hum Reprod. 1999;14:2838-43. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology. 1987;24:387-402. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature. 1985;313:573-5. Ramezani M, Valojerdi MR, Parivar K. Effect of three vitrification methods on development of two-cell mouse embryos. Cryo Letters. 2005;26:85-92. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:184-91. Sohn IP, Ahn HJ, Park DW, Gye MC, Jo DH, Kim SY, Min CK, Kwon HC. Amelioration of mitochondrial dysfunction and apoptosis of two-cell mouse embryos after freezing and thawing by the high frequency liquid nitrogen infusion. Mol Cells. 2002;13:272-80. Stowinska M, Karol H, Ciereszko A. Comet assay of fresh and cryopreserved bull spermatozoa. Cryobiology. 2008;56:100-2. Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano A. Assessment of DNA damage in individual hamster embryos by comet assay. Mol Reprod Dev. 1999;54:1-7. Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz RM, Okano A. Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay. Theriogenology. 2000;54:13745. Vajta G, Booth PJ, Holm P, Greve T, Callesen H. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo-Letters. 1997;18:191-5. Yeoman RR, Gerami-Naini B, Mitalipov S, Nusser KD, WidmannBrowning AA, Wolf DP. Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts. Hum Reprod. 2001;16:1965-9. Yokota Y, Sato S, Yokota M, Yokota H, Araki Y. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril. 2001;75:1027-9.

References

Ali J, Shelton J. Development of vitrification solutions. In: Vitrification in assisted reproduction: A user’s manual and trouble-shooting guide. Editors Tucker MJ, Liebermann J. Informa Healthcare, London. 2007. Chung JT, Tosca L, Huang TH, Xu L, Niwa K, Chian RC. Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development. RBM Online. 2007;15:198-207.

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Liability Risks Associated with Mismanagement of Sperm Specimens Fethiye Sinem Karipcin, MD; Amjad Hossain, Ph.D.; John Y. Phelps, MD, JD, LLM Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, The University of Texas Medical Branch, 301 University Blvd. Galveston, TX 77555 USA Corresponding author: Fethiye Sinem Karipcin, MD Department of Obstetrics & Gynecology, The University of Texas Medical Branch, 301 University Blvd. • Galveston, TX 77555-0587 Email: fskaripc@utmb.edu • Tel: 409-772-2999 • Fax: 409-747-0366 Introduction

male gamete, testicular cancer, fertility clinic, wrong sperm, cryopreservation, IVF, ART,and storage.

Laboratory personnel working in fertility clinics need to be diligent in their management of sperm specimens. Laxity in record keeping, failure to properly maintain sperm specimen, inattentiveness, and misconduct by laboratory personnel unnecessarily exposes fertility clinics and their staffs to potential litigation. A single lawsuit for mismanagement of sperm specimens can not only be financially devastating, but can ruin the careers of all those involved. In addition to civil monetary actions by plaintiff attorneys, mismanagement of sperm specimens can lead to loss of licensure and credentialing of fertility laboratories and their staffs. In this article, we review prior legal cases arising from mismanagement of sperm specimens in fertility laboratories. By drawing attention to those legal cases, we emphasize the importance of being meticulous in all aspects of sperm management including collection, preparation, record keeping, cryopreservation, and insemination, not only to provide quality patient care, but also to avoid exposure to potential litigation.

Misconduct by fertility clinic personnel

One of the more famous cases occurred in 1992 when a physician used his own sperm to inseminate patients who were seeking infertility treatment from his clinic. In this case, Dr. Cecil B. Jacobson, a fertility specialist in a Virginia-based clinic in Vienna, Virginia , Reproductive Genetics Center, faced criminal and civil liability when he used his own sperm for insemination. Dr. Jacobson was convicted of 52 felony counts for the gross ethical and legal code violation in handling reproductive tissues (1). Dr. Jacobson was sentenced to 5 years in prison, to be followed by 3 years of probation, and ordered to pay $114,205 in fines and restitution. Jacobson, suspected of being the father of 75 fertility babies, inseminated women with his own sperm instead of donor sperm without their consent. ASRM guidelines for selection of sperm donors restricts the owner, operator, laboratory director, or employees of the facility performing therapeutic donor insemination (TDI) to serve as donors in that practice (2–4). Therefore, even if the patient desires to be inseminated by a member of fertility clinic personnel, her wish can’t be followed according to ASRM guidelines. Owners and laboratory directors need to be very careful in selecting laboratory personnel who directly handle sperm specimens and avoid hiring employees with prior histories of criminal, psychiatric, or drug problems. Also, insurance companies providing liability coverage for fertility clinics are unlikely to cover lawsuits arising from insemination with sperm from laboratory personnel since this is an intentional act and insurance companies routinely provide coverage for negligent acts only, rarely providing coverage for intentional acts or acts that are outside the scope of employment. The

Materials and Methods

Legal cases, pertaining to mishandling of sperm were obtained using Westlaw and LexisNexis legal search engines. Westlaw and LexisNexis are the 2 primary commercial online legal research engines for legalrelated materials. The online legal research engines and services provided by Westlaw and LexisNexis are used mostly by attorneys or law students. Legal research engines are important in the legal profession to reinforce legal arguments. The United States legal system operates under the principle of stare decisis, which is a system of legal precedents to ensure that the courts deliver consistent rulings on similar legal issues. Thus, prior rulings are helpful to courts in deciding cases. In preparation of this article, the following keywords were used in the search: reproduction, semen,

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financial burden for defending the lawsuit and paying for any monetary penalties awarded during the course of litigation will most likely entirely fall on the fertility clinic and not on the insurance provider.

careful with these specimens since the source of sperm is limited. Unlike specimens received from men who retain the ability to produce sperm, any mismanagement of these specimens may result in complete loss of the male partner’s ability to produce genetic offspring in the future. Consequently, these mistakes are more likely to lead to litigation with higher monetary awards since the harm is irreparable. Similarly, laboratory personnel need to be especially careful with sperm specimen acquired by testicular biopsy or epididymal sperm aspiration since loss of these specimens will necessitate another surgical procedure to obtain sperm. In our laboratory, we maintain specimens obtained from men who have either lost the ability to produce sperm or require surgical intervention to obtain sperm separate from the more “routine” specimens obtained from men who do not have such restrictions. We also use highlighted warning stickers to distinguish these specimens from the more “routine” specimens (7,8). In a Georgia case, the Atlanta Center for Reproductive Medicine was sued for professional negligence, breach of contract, negligent misrepresentation, negligent infliction of emotional distress, unnecessary medical procedures, and breach of fiduciary duty after they used all of a cancer patient’s cryopreserved sperm in one IVF cycle (9). The cancer patient had stored his sperm at the Atlanta Center for Reproductive Medicine before undergoing cancer treatment. After the first IVF cycle was unsuccessful, his wife started a second IVF cycle with the understanding there was enough of her husband’s sperm remaining. The second IVF cycle then had to be cancelled after it was discovered there was no remaining sperm because all the cryopreserved sperm was thawed and used for the first IVF cycle. This illustrates the importance of proper accounting of cryopreserved sperm specimens and the need for communication between the reproductive endocrinologist and laboratory personnel. Complexity in record keeping can be further compounded by change in ownership of a laboratory and change in personnel that expectedly occurs as a clinic ages. Some fertility clinics in the United States are more than 20 years old. During this 20-year period, ownership of a clinic may have changed, and even if the owner remains the same, it is highly likely that multiple changes have occurred in the laboratory staff responsible for maintaining cryopreserved specimens. Any time there is a change in ownership or laboratory personnel, an inventory of cryopreserved specimens should be performed. Also, a partial solution may be to charge an annual storage fee for

Mistakes in record keeping and labeling of sperm Diligence in record keeping and labeling is an absolute must for all laboratory and clinical personnel involved in handling sperm. Without proper record keeping, loss, destruction, and commingling of samples is far more likely. There are numerous cases that arise from lack of diligence in record keeping and labeling. In Hamicher v. University of Utah Medical Center, the university fertility clinic was sued because the wrong donor sperm was used for IVF that produced triplets (5). This mistake arose from failure to properly maintain the cryopreservation records. In a New York case, Skolnick v. Idant Corporation, Advanced Fertility Services, and Hugh D. Melnick, MD, a white woman was supposedly inseminated by her white husband’s sperm. The 24-year-old husband was terminally ill and had his sperm cryopreserved before undergoing treatment for Ewing sarcoma. Prior to the husband’s death, the wife gave birth to a black girl (6). The wife then sued the fertility clinic for malpractice, negligence, fraud, battery, loss of opportunity to have her husband’s child, breach of privilege, and breach of contract seeking $50 million in compensation. Prior to going to trial, the lawsuit settled for $400,000. Mislabeling of sperm of a man scheduled to receive chemo/radiotherapy—for whom sperm cryopreservation is the only way to have a biologic child—is an irreparable mistake. Laboratory personnel handling deposited sperm specimens produced from men prior to cancer treatment, vasectomy, or surgical therapies need to be especially

Note to Advertisers Please request 2010 advertising rates for both hardcopy advertising and website banner ads. A discount of 10% is available with a prepaid agreement covering four (4) consecutive issues. For more information, please contact The Journal of Clinical Embryology™ advertising office: jceadvertising@bellsouth.net • (352) 331-5235 — The Publisher

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cryopreserved specimes. By charging an annual storage fee, there is at least a financial incentive to perform an accounting of the cryoperserved specimens each year.

in laws that explicitly state the time period after which a fertility clinic can dispose of sperm from patients. For this reason, the best resource for guidance if contemplating destroying cryospreserved sperm is the original contract signed by the patient and the clinic at the time the sperm specimen was obtained. If a dispute arises, courts will first look at the contract in deciding liability for disposing of sperm. To avoid future disputes, fertility clinics should review and update their current contracts if there is any ambiguity about how long a fertility clinic will cryopreserve sperm prior to disposing of it. It is undoubtedly better to address this complicated issue of sperm disposal in the original contract between the patient and the fertility clinic in plain and clear language rather than have it disputed later. It would be unlikely for a patient to prevail in a lawsuit against a fertility clinic for disposing of sperm if the original contract was clear and concise on the issue of when the fertility clinic can dispose of the sperm. To protect the interest of the fertility clinic, the contract should have provisions stating the fertility clinic has the right to dispose of the cryoperserved sperm after a set period of time. By charging patients an annual storage fee, the issue of what is a reasonable amount of time for a fertility clinic to store sperm prior to disposing of it may be easier to resolve. For example, the contract could state in clear language that if the patient is more than 2 years delinquent in paying the annual storage fee, the fertility clinic will dispose of the sperm. If the fertility clinic is not charging an annual storage fee, the contract should have explicit provisions stating the specific time period of cryopreservation before the sperm may be destroyed. If there was no contract between the patient and the fertility clinic or if the contract failed to have provisions for disposing of sperm, the issue of what is a reasonable amount of time to cryopreserve sperm prior to disposing of it becomes more complex. What constitutes a reasonable amount of time is debatable. Regardless of contract provisions, it is not only courteous, but in the legal interest of the fertility clinic to send a letter by certified mail to the patient’s last known address putting them on notice their sperm will be disposed of after a certain amount of time and giving the patient the option to renew their contract or pick up their specimens. The combination of having explicit provisions in the original contract for disposing of sperm and sending patients a certified notice letter will make it difficult for a patient to later prevail in a lawsuit against a fertility clinic for deposing of cryopreseved sperm.

Failure to maintain storage tanks

Failure to maintain storage tanks is another source of exposure to litigation for fertility clinics. The South Florida Institute for Reproductive Medicine was sued when the sperm cryopreservation storage tank’s cooling apparatus failed, leading to destruction of cryopreserved sperm samples (10). The plaintiff in this case, Mr. Kurchner, was diagnosed with cancer and deposited 5 samples of sperm with the fertility laboratory for cryopreservation prior to undergoing chemotherapy. For those specimens obtained from men who do not retain their ability to produce specimens as a result of cancer treatment or who need surgical intervention to obtain sperm, special consideration should be given to storing these specimens in 2 different storage tanks and in different locations. By having a back-up specimen stored in a different location, it is less likely a man’s ability to produce genetic offspring will be forever lost. Nonetheless, there is no guarantee that cryoperserved specimens will not be destroyed by fire, flood, or natural disaster (7,11,12). Storage practices related to the preservation unit of a fertility clinic are uniquely different from that of other activities of the clinic. Storage tanks require continuous servicing for proper maintenance of preserved sperm. For all laboratory procedures except cryo, there is a finish line. In a sense, there is no such finish line in a cryo procedure until the sample is utilized or disposed of, which can be years away. A storage tank, once in use, is never completely empty; some sperm samples are always there. Thus, the responsibility of maintaining the tank never ends, and accordingly, the commitment of laboratory personnel to the preservation procedure is uniquely demanding. Any negligence in filling up a cryopreservation tank with liquid nitrogen can influence the quality of the cryo sperm or may cause total loss of the specimen pool (7).

Disposing of cryopreserved sperm

Disposing of stored samples by the fertility clinics can be very difficult and complicated. The fertility clinic at the University of Texas Health Science Center at Dallas was sued for disposing of sperm without notifying the patient who had cryopreserved his sperm at the fertility clinic prior to being treated for testicular cancer (13). The fertility clinic at the University of Texas Health Science Center stored the sperm for 8 years prior to disposing of it. There is a paucity

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Other possible scenarios that might result in litigation

We recommend the following to deter litigation: • Careful selection and screening of the laboratory personnel • Special handling and separate storage of deposited sperm specimens acquired by testicular biopsy and epididymal sperm aspiration, and sperm specimens produced from men prior to cancer treatment , vasectomy, surgical therapies • Good communication between laboratory personnel and the fertility specialist • Diligent record keeping •A yearly inventory of cryopreserved sperm specimens, which should be repeated earlier anytime there is a change in ownership or laboratory personnel • Have patients sign a contract that has explicit provisions for disposal of sperm specimens for delinquency in paying annual storage fee. • Separate storage tanks for sperm specimens from HIV, Hepatitis C and B positive patients • Preparedness for natural disasters for clinics located in areas at risks for earthquakes, hurricanes and floods. n

Transmission of infectious disease to laboratory employees or to patients is another potential source of litigation. Special care should be taken to ensure lab personnel are following universal precautions. There are reports of cross-contamination with HIV in nitrogen storage tanks(14). For this reason it is recommended cryopreserved specimens from patients with HIV and/ or Hepatitis be stored in separate tanks to minimize the risk of cross contamination during storage. Natural disasters such as earthquakes, hurricanes and floods as well as fires can result is destruction of cyropreserved specimens. For laboratories located in areas at high risk for earthquakes, hurricanes or floods consideration should be given to long term storage of specimens in another facility. However, transportation and storage in another building may significantly increase cost.

Regulation of Fertility Clinics

Governmental oversight of clinical laboratories including fertility clinic activities falls under CLIA and FDA regulations (CLIA: www.cdc.gov/clia/regs/toc.aspx FDA: www.fda.gov/cber/gdlns/tissue2.txt, www.fda.gov/cber/ tissue/docs.htm;). Regulatory compliance of fertility clinics is also monitored by governmental agencies including both federal and state governments and by private organizations such as the College of American Pathologists (CAP: www. cap.org), and the Joint Commission of the Accreditation of Healthcare Organizations (JCAHO: www.jcaho.org) . Some of these oversight agencies such as the American Association of Tissue Banks (AATB: www.aatb.org), have standards that are more stringent than federal standards. The American association for reproductive medicine (ASRM: www.asrm.org) also has useful guidelines related to cryopreservation for fertility treatment. The goal of all these regulatory and monitoring agencies is to ensure quality laboratory services in the fertility clinics.

References

1. St. Paul Fire & Marine Insurance Company v. Jacobson, 48 F.3d 778; US App. Lexis 3089, 1995. 2. The practice committees of ASRM. Compendium. Fertil Steril., 2008;90:S30–S44. 3. The practice committees of ASRM. Essential elements of informed consent for elective oocyte cryopreservation: a Practice Committee opinion. Fertil Steril. 2008;90:S134–5. 4. The ethics committee of ASRM. Fertility preservation and reproduction in cancer patients. Fertil Steril. 2005;83:1622–8. 5. Harnicher v. University of Utah Medical Center, 962 P.2d 67; 349 Utah Adv. Rep. 3; Lexis 57, 1998. 6. Penofsky DJ. Liability of Sperm Banks. Am Jur Trials on Westlaw [serial online]. 2005;50:1–269. 7. Hossain A, Nagamani M. Cryopreservation of Male Gamets. In Botros R, Juan GV, Antonis M eds. Infertility and Assisted Reproduction. New York: Cambridge University Press; 2008; 466–478. 8. Phelps JY. Restricting access of human immunodeficiency virus (HIV)-seropositive patients to infertility services: a legal analysis of the rights of reproductive endocrinologists and HIV-seropositive patients. Fertil Steril. 2007;88:1483–90. 9. Baskette v. Atlanta Center for Reproductive Medicine, 285 Ga. App. 876; SE 2d 100; Lexis 103, 2007. 10. Kurchner v. State Farm Fire and Casualty Co., 858 So. 2d 1220; Fla App. Lexis 17096, 2003. 11. Centola GM. The Art of donor gamete cryobanking: current considerations. J Androl. 2002;3:174–179. 12. Crister Jk. Current status of semen banking in the USA. Human Reprod. 1998;13(suppl):55–67. 13. Stanton v. University of Texas Health Sciences Center at Dallas, 997 SW 2d 628; Tex. App. Lexis 7142, 1998. 14. Clark GN. Sperm cryopreservation: is there a significant risk of cross contamination? Hum Reprod 1999;14:2941–3

Summary

Fertility laboratories can be a target for an array of adverse actions including law suits, fines, loss of licensure, and certification for mismanagement of sperm specimens. The law cases in this article illustrate the high diligence required in every step of handling the sperm including collection, labeling, preparation, record keeping, maintenance of cryopreservation facilities, and overseeing the integrity and competence of laboratory personnel. By exercising diligence at each step, laboratories can lessen their exposure to potential litigation.

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Meetings for the Embryologist (Please send announcements for meetings you would like to see included in this listing)

The American Society For Cell Biology Annual Meeting 05-Dec-2009 - 09-Dec-2009 San Diego CA USA As the premier international meeting in the field of cell biology, the ASCB Annual Meeting is intended for scientists and students in academia, industry, government, and higher education. Over 100 scientific sessions and 3,500 poster presentations cover a variety of scientific areas within the discipline. With opportunities to learn about the latest research and network with peers, the ASCB Annual Meeting appeals to the diverse interests of the international cell biology community. Attendees view the ASCB Annual Meeting as the place to: • • • • • • • • • • •

Hear and discuss the most exciting research discoveries in cell biology in detail Gain a broad view of research in the field Network with senior scientists and others See friends and colleagues Gain valuable career development advice/knowledge Visit over 350 commercial and nonprofit exhibits offering new products and services Look for a job, fellowship, postdoctoral position, or new collaborator Improve teaching knowledge and skills Recruit and/or interview job candidates Attend sessions of special interest to students, minorities and women Learn where and how to apply, and advocate for, research funding Registration Deadline: 01-Oct-2009 http://www.ascb.org/meetings/

Frontiers in Reproductive Biology and Regulation of Fertility 01-Feb-2009 - 05-Feb-2009 Santa Fe New Mexico USA “Sexual reproduction is a complex and highly regulated process essential for successful propagation and diversification of genetic complements, beginning with germ cell development and completing with birth of live young. For successful reproduction, a plethora of events including gametogenesis, ovulation, fertilization, implantation, and placentation requires precise regulation; the success of each go-ahead event is dependent on the accomplishment of the preceding event. Research on these events and their coordination has been translated into clinical practice, particularly for enhancing successes in fertility clinics, controlling gynecological diseases and developing contraceptives. This meeting is designed to bring together a diverse group of leaders, established and rising in the field, who study the complex regulation of reproduction and related developmental processes. The speakers and participants will comprise a group that uses a variety of model systems to better understand the processes relevant to human and animal reproduction and fertility regulation. “ Registration Deadline: 30-Oct-2008 http://www.keystonesymposia.org/Meetings/ViewMeetings.cfm?Meetin

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4th International Symposium on the Genetics of Male Infertility February 4-6,2010, Salt Lake City, UT The 2010 meeting will be held at the Canyons ski resort in Park City in early February. Thank you for your interest in the Genetics of Male Infertility International Symposium. http://medicine.utah.edu/malegenetics/ International Symposium Information Andrology Department University of Utah School of Medicine 675 Arapeen Drive Suite 205, Salt Lake City, UT 84108 Phone: 801-581-3740 Fax: 801-581-6127 Email: malegeneticsinfo@hsc.utah.edu

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 Barry Behr, PhD, HCLD Director, IVF/ART Laboratory Co-Director, Stanford Fertility and Reproductive Medicine Centers REI/IVF Program Associate Professor, Department of OB/GYN Behr@Stanford.EDU

Carol Brenner, PhD Departments of Obstetrics & Gynecology and Physiology Wayne State University School of Medicine Detroit MI 48201 cbrenner@med.wayne.edu Grace Centola, Ph.D., H.C.L.D. Professional ReproLab Consulting Macedon, New York centolag@yahoo.com 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 David Mortimer, Ph.D. President, Oozoa Biomedical Box 93012 Caulfeild Village RPO, West Vancouver, BC, V7W 3G4 Canada david@oozoa.com

Thomas B. Pool, Ph.D. HCLD Fertility Center of San Antonio San Antonio, TX rpool@fertilitysa.com 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 Alan Thornhill, Ph.D. HCLD Scientific Director The London Bridge Fertility, Gynaecology and Genetics Centre One St Thomas Street, London Bridge, London SE1 9RY England athornhill@thebridgecentre.co.uk 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

Ganirelix Acetate Injection BRIEF SUMMARY (For full Prescribing Information, see package insert.)

Geriatric Use Clinical studies with Ganirelix Acetate Injection did not include a sufficient number of subjects aged 65 and over.

INDICATIONS AND USAGE Ganirelix Acetate Injection is indicated for the inhibition of premature LH surges in women undergoing controlled ovarian hyperstimulation. CONTRAINDICATIONS Ganirelix Acetate Injection is contraindicated under the following conditions: • Known hypersensitivity to Ganirelix Acetate or to any of its components. • Known hypersensitivity to GnRH or any other GnRH analog. • Known or suspected pregnancy (see PRECAUTIONS). WARNINGS Ganirelix Acetate Injection should be prescribed by physicians who are experienced in infertility treatment. Before starting treatment with Ganirelix Acetate, pregnancy must be excluded. Safe use of Ganirelix Acetate during pregnancy has not been established (see CONTRAINDICATIONS and PRECAUTIONS). PRECAUTIONS General Cases of hypersensitivity reactions, including anaphylactoid reactions with the first dose, have been reported during post-marketing surveillance (see ADVERSE REACTIONS). The packaging of this product contains natural rubber latex which may cause allergic reactions. Information for Patients Prior to therapy with Ganirelix Acetate Injection, patients should be informed of the duration of treatment and monitoring procedures that will be required. The risk of possible adverse reactions should be discussed (see ADVERSE REACTIONS). Ganirelix Acetate should not be prescribed if the patient is pregnant. Laboratory Tests A neutrophil count ≥ 8.3 ( x 109/L) was noted in 11.9% (up to 16.8 x 109/L) of all subjects treated within the adequate and well-controlled clinical trials. In addition, downward shifts within the Ganirelix Acetate Injection group were observed for hematocrit and total bilirubin. The clinical significance of these findings was not determined. Drug Interactions No formal drug-drug interaction studies have been performed. Carcinogenesis and Mutagenesis, Impairment of Fertility Long-term toxicity studies in animals have not been performed with Ganirelix Acetate Injection to evaluate the carcinogenic potential of the drug. Ganirelix Acetate did not induce a mutagenic response in the Ames test (S. typhimurium and E. coli) or produce chromosomal aberrations in in vitro assay using Chinese Hamster Ovary cells. Pregnancy Pregnancy Category X Ganirelix Acetate Injection is contraindicated in pregnant women. When administered from Day 7 to near term to pregnant rats and rabbits at doses up to 10 and 30 μg/day (approximately 0.4 to 3.2 times the human dose based on body surface area), Ganirelix Acetate increased the incidence of litter resorption. There was no increase in fetal abnormalities. No treatment-related changes in fertility, physical, or behavioral characteristics were observed in the offspring of female rats treated with Ganirelix Acetate during pregnancy and lactation. The effects on fetal resorption are logical consequences of the alteration in hormonal levels brought about by the antigonadotrophic properties of this drug and could result in fetal loss in humans. Therefore, this drug should not be used in pregnant women (see CONTRAINDICATIONS). Nursing Mothers Ganirelix Acetate Injection should not be used by lactating women. It is not known whether this drug is excreted in human milk.

ADVERSE REACTIONS The safety of Ganirelix Acetate Injection was evaluated in two randomized, parallel-group, multicenter controlled clinical studies. Treatment duration for Ganirelix Acetate ranged from 1 to 14 days. Table IV represents adverse events (AEs) from first day of Ganirelix Acetate administration until confirmation of pregnancy by ultrasound at an incidence of ≥ 1% in Ganirelix Acetate-treated subjects without regard to causality. TABLE IV: Incidence of common adverse events (Incidence ≥ 1% in Ganirelix Acetate-treated subjects). Completed controlled clinical studies (All-subjects-treated group). Adverse Events Occurring in ≥ 1% Abdominal Pain (gynecological) Death Fetal Headache Ovarian Hyperstimulation Syndrome Vaginal Bleeding Injection Site Reaction Nausea Abdominal Pain (gastrointestinal)

Ganirelix Acetate N=794 % (n) 4.8 (38) 3.7 (29) 3.0 (24) 2.4 (19) 1.8 (14) 1.1 (9) 1.1 (9) 1.0 (8)

During post-marketing surveillance, rare cases of hypersensitivity reactions, including anaphylactoid reactions with the first dose, have been reported (see PRECAUTIONS). Congenital Anomalies Ongoing clinical follow-up studies of 283 newborns of women administered Ganirelix Acetate Injection were reviewed. There were three neonates with major congenital anomalies and 18 neonates with minor congenital anomalies. The major congenital anomalies were: hydrocephalus/meningocele, omphalocele, and Beckwith-Wiedemann Syndrome. The minor congenital anomalies were: nevus, skin tags, sacral sinus, hemangioma, torticollis/asymmetric skull, talipes, supernumerary digit finger, hip subluxation, torticollis/high palate, occiput/abnormal hand crease, hernia umbilicalis, hernia inguinalis, hydrocele, undescended testis, and hydronephrosis. The causal relationship between these congenital anomalies and Ganirelix Acetate is unknown. Multiple factors, genetic and others (including, but not limited to ICSI, IVF, gonadotropins, progesterone) may confound ART (Assisted Reproductive Technology) procedures. OVERDOSAGE There have been no reports of overdosage with Ganirelix Acetate Injection in humans.

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 76847 7/08 12-JBS

Short and Simple Short: Mean duration of treatment — 5.4 days1* Simple: Only GnRH antagonist available in a ready-to-use pre-filled syringe1

✔ Rapidly absorbed - approximately 1 hour after dosing1

✔ Rapidly reversible - within 48 hours of

discontinuation, pituitary hormones (LH and FSH) are fully recovered1

✔ Small injection volume – 0.5 mL

✔ Room temperature storage

*Results from a multicenter, open-label randomized study to assess the efficacy and safety of Ganirelix Acetate Injection in women undergoing controlled ovarian hyperstimulation. Range 2-14 days.

Ganirelix Acetate Injection is indicated for the inhibition of premature LH surges in women undergoing controlled ovarian hyperstimulation.

IMPORTANT SAFETY INFORMATION

Ganirelix Acetate Injection is contraindicated in patients with a known hypersensitivity to ganirelix acetate or to any of its components, to GnRH or any other GnRH analog, and in patients with known or suspected pregnancy. Only physicians experienced in infertility treatment should prescribe Ganirelix Acetate Injection. Before starting treatment with ganirelix, pregnancy must be excluded. Cases of hypersensitivity reactions, including anaphylactoid reactions with the first dose, have been reported during post-marketing surveillance. The packaging of this product contains natural rubber latex which may cause allergic reactions. The most common adverse events occurring in ≥1% of patients treated with ganirelix in clinical trials (N=794) include: abdominal pain (gynecological) 4.8%, fetal death 3.7%, headache 3.0%, ovarian hyperstimulation syndrome (OHSS) 2.4%, vaginal bleeding 1.8%, injection site reaction 1.1%, nausea 1.1%, abdominal pain (gastrointestinal) 1.0%. Please see adjacent page for brief summary of full Prescribing Information. Reference: 1. Ganirelix Acetate Injection [package insert]. Roseland NJ: Organon USA Inc. Copyright © 2009, Schering Corporation, Kenilworth, NJ 07033. All rights reserved.

Leaders in the Field of Fertility Printed in the USA.

GX2036

10/09