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Age and Fertility Your Biological Clock Is TICKING! As you grow older your chances of conceiving decrease. This begins at age 25. FertilageTM for Women was developed to help slow down the clock for aging of your reproductive system. Key ingredients were selected to help you. FertileAgeTM for men was designed to help men remain verile as they age. FertileAge.com has fertility supplements for both MEN and WOMEN to help you throughout your child-bearing years. Go to www.FertileAge.com for all your fertility supplement needs. Order Online at www.FertileAge.com US/Canada: 800-720-6375 ©2005 FertileAge.com • International: 519-826-5800 • Fax: 519-826-6947 • email: [email protected] Age-2005-05 CONTENTS Fertility Magazine The First Magazine In FertilityTM FEATURED ON THE COVER Pictured on the front cover of Fertility Magazine, Volume 4 is Lauren Cecchi. The Challenge and Promise of Blastocyst Cryopreservation by Marius Meintjes, PhD . . . . . . . . 11 The Effect of CO 2 Concentration and pH on the In-Vitro Development of Mouse Embryos by Avner Hershlag, MD and Huai L. Feng, PhD, HCLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 An Overview of the Effects of Age on Fertility in Women by Don Rieger, PhD . . . . . . . . . . . . . . 31 23 Degree of Re-expansion of Thawed Blastocysts is Predictive of Frozen Embryo Transfer (FET) Success 41 FEATURES 8 Preimplantation Genetic Diagnosis: A Domestic Animal Perspective by W. Allan King, PhD 15 Use of the Sperm Chromatin Structure Assay (SCSA®) as a Diagnostic Tool in the Human Infertility Clinic by Donald P. Evenson, PhD, HCLD and Regina L. Wixon, PhD 23 Degree of Re-expansion of Thawed Blastocysts is Predictive of Frozen Embryo Transfer (FET) Success by Deborah C. Merryman, MSc, ELD/TS (ABB) 27 Advanced Maternal Age and Infertility by Alan B. Copperman, MD 29 ART and Aging by Michael S. Neal, BSc, MSc, PhD candidate Patient’s Corner 41 Patient’s Corner by Charlene Alouf, PhD and Albert El-Roeiy, MD, MBA ©2005 IVFonline 4 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG 45 Helping You Understand “What Happens Next?” by Liz Sanders Helping You Understand “What Happens Next?” 46 Feature Products 47 Hypnosis for Childbirth by Shawn Gallagher, BA, CHt 49 Human Resources 45 50 Resources 46 Oocyte Cryopreservation: A Common Sense Approach by James Stachecki, PhD CONTRIBUTORS Marius Meintjes, PhD Avner Hershlag, MD Don Rieger, PhD Huai L. Feng, PhD, HCLD W. Allan King, PhD Donald P. Evenson, PhD, HCLD Regina L. Wixon, PhD Deborah C. Merryman, MSc, ELD/TS (ABB) Editor in Chief: Monica Mezezi, MBA Design: Debbie Nagy Editorial Office: IVFonline, 24 Norwich St. E. Guelph, Ontario, Canada, N1H 2G6 US/Canada: 800-720-6375 International: 519-826-5800 Fax: 519-826-6947 Email: [email protected] www.IVFonline.com www.FertilityMagazine.org Alan B. Copperman, MD Michael S. Neal, BSc, MSc, PhD candidate Charlene Alouf, PhD Albert El-Roeiy, MD, MBA Liz Sanders Shawn Gallagher, BA, CHt James Stachecki, PhD Instructions to Contributors To submit an Article/Abstract email us at: [email protected] To submit an Ad email us at: [email protected] Fertility Magazine and all its associates ©2004, All Rights Reserved. Covers, contents, images, ads in print or web form are copryight protected and reprinting or reproduction of any kind is expressly prohibited without the written permission of Fertility Magazine. Fertility Magazine does not knowingly accept false or misleading advertising, articles, opinions or editorial, nor does the publisher assume any responsibility for the consequences that occur should any such material appear, and assumes no responsibility for content, text, opinions or artwork of advertisements appearing in Fertility Magazine in print or web form. Some of the views expressed by contributors may not be the representative views of the publisher. WWW.FERTILITY MAGAZINE .ORG • VOLUME 4 • FERTILITY MAGAZINE 5 FREE SUBSCRIPTION Free Subscription… Subscribe today to Fertility Magazine …”The First Magazine In FertilityTM ” By joining us today you will receive the latest in: • International News • Scientific information• Patient Corner • Alternative Medicine All the information needed to keep you up-to-date in Fertility. Join Now …..Join Today Call US/Canada: 800-720-6375, International: 519-826-5800, email: [email protected], or Fax the form below to: 519-826-6947. Yes …Sign me up! To receive Fertility Magazine …”The First Magazine In FertilityTM” Name: Address: City: State: Email address: Send it to a Friend at email: Zip Code: Country: INTERNATIONAL NEWS Preimplantation Genetic Diagnosis: A Domestic Animal Perspective by W. Allan King, PhD Professor and Canada Research Chair, Department of Biomedical Sciences University of Guelph, Guelph, ON Canada, N1G 2W1 P reimplantation genetic diagnosis (PGD) is a technique born out of the desire of livestock breeders to intensify the accuracy of selection of their livestock, and the desire of parents to ensure the health of their children. The technique is the culmination of scientific discoveries over a period of time spanning Mendel's postulations on the principles of inheritance in 1866 and Mullis's development of polymerase chair reaction (PCR) in Research Institute in Ottawa reported the successful application of the approach to another species. This time, 2 week-old cattle embryos were sexed by chromosome analysis of trophoblast cells. The procedure had limitations, mainly based on the number of metaphases required to make an accurate diagnosis and the need to use embryos of later stages in order to provide enough cells without compromising the longterm viability of the embryo. With The application of PGD is increasing on an almost daily basis. 1983. Genetic screening of preimplantation embryos was first documented in a paper by Gardner and Edwards at Cambridge University in the April 1968 issue of Nature. The abstract of the paper stated that “An important step forward has been made in controlling the sex of rabbits by sexing pre-implantation embryos and transferring the sexed embryos into recipient females. In the experiments, fetuses were surgically delivered and their sex was found to have been correctly predicted at the blastocyst stage.” The procedure consisted of piercing the mucin coat and zona pellucida of day 5 blastocysts and removing 200 to 300 blastomeres, which were fixed and stained to reveal the presence or absence of the Barr body. It was another 8 years before Hare and colleagues at the Animal Disease 8 FERTILITY MAGAZINE • VOLUME 4 • the potential and proof of the principle clearly established, the pressure was on to stream-line the technique, and make it compatible with the commercial practice of using day-7 cattle blastocysts for cryopreservation and transfer. The ground-breaking work on embryo bisection by Willadsen at Cambridge in 1979 led the way to embryo splitting and biopsy, which quickly became a commercially viable option for increasing the rates of pregnancy and twinning in valuable cattle. It soon became apparent that if a cattle blastocyst could be split in half without severely compromising the developmental potential of either half, one demi-embryo could be subjected to chromosome analysis and the other half frozen or transferred. In 1985, Picard and colleagues at the University of Montreal used this approach and WWW.FERTILITYMAGAZINE.ORG W. ALLAN KING, PHD reported the birth of calves from frozen, sexed embryos. While somewhat successful, the technique was far from suitable for routine application in commercial settings because of the need for sophisticated microscopes, trained technologists and, more importantly, adequate number of metaphase spreads for analysis to rule out diagnostic errors since blastocysts had been shown to contain subpopulations of aneuploid and polyploid cells. The advent and commercialization of the analysis of DNA by PCR technology created new vistas in the genetic analysis of single cells. By the end of the 1980's, cattle embryos were being routinely sexed, often on the farm, by PCR amplification and analysis of sex chromosome linked genes. Around this time, reports on the analysis of human embryo biopsies also started to appear in the literature. The birth of children subsequent to genetic screening of embryos was reported INTERNATIONAL NEWS in 1994. The application of PGD is increasing on an almost daily basis. In domestic animals, there are three main areas of application: sex selection, avoidance of genetic defects and selection of production characteristics. In agricultural practices, one sex is often favoured over the other. For example, the artificial insemination industry is based on the dissemination of semen from the elite sons of animals of high genetic merit, while milk production requires high producing females. In addition, a number of genetic defects including bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS), have been identified in cattle for which DNA probes are commercially available. In a PGD context this offers the breeder a unique opportunity to pre-select the offspring rather than culling individuals postnatally. Finally, since most production traits in domestic animals are quantitative genetic traits controlled by numerous genes, intensive research into developing markers for such traits as milk yield and composition, quality of meat and disease resistance is in progress. Several markers based on single nucleotide polymorphisms and micro-satellite technologies, have already been identified in dairy cattle for traits including milk production and resistance to mastitis. The use of such markers for marker assisted selection (MAS), in combination with embryo biopsy and embryo transfer techniques, offers the means to realizing the mission of ensuring the accuracy of selection of breeding livestock and maximizing the efficiency of production. PGD has come a long way since its initiation and become an important tool for agriculture industries. The potential and practice of this approach were barely imagined 40 years ago when the first commercial embryo transfers were performed in cattle. One can only wonder if Mendel had any inkling that his musings on factorial inheritance in peas would one day lead scientists to predict the sex and genetic status of the conceptus and help breeders pre-select their animals for production traits. You can contact W. Allan King, PhD at: [email protected] WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 9 global® culture media was scientifically designed with the embryo in mind global® is a single culture medium used to culture embryos from Day 1 to blastocyst stage. global®’s development began with the work of John Biggers. John Biggers and his development team used the principles of Simplex Optimization to develop the medium for mice, it was then adapted into a human embryo culture media through the efforts of our scientific committee lead by Klaus Wiemer, PhD. global ® culture media has been proven in multiple independent centers worldwide to help increase the number of embryos developing to either Day 3 culture or blastocyst culture , yielding more embryos available for transfers and has shown significant increased pregnancy rates. The Media IVF patients have greatly benefited from the use of global®, as it has been shown to increase pregnancy results. Fertility clinics who have adopted the use of global® have reported improved embryonic development, blastocyst development and an increase in the number of embryos available for use in transfer or for cryopreservation. The Patients global ® has proven to be stable in its use, maintains its pH levels, Osmolality and has shown to produce the lowest levels of ammonia build-up over time. Superior stability, quality and superior performance. The Embryos LifeGlobal®, ‘The Art Media Company’ was established to meet the industry needs for high quality, specially designed media products. All products are designed with the nutritional needs of the human embryo in mind, and with the strictest quality controls, tested ingredients, each batch is independently tested and are FDA compliant. Our research team of John Biggers, Jacques Cohen, Klaus Wiemer, and Don Rieger and their years of research and experience have developed these products, offering you the best quality and performance. LifeGlobal has a well established worldwide distribution network to assure you quality, customer service, and fresh delivery of the products. www.LifeGlobal.com Order Online at www.IVFonline.com US/Canada: 800-720-6375 • International: 519-826-5800 • Fax: 519-826-6947 • email: [email protected] ©2005 LifeGlobal ® LG-global-2005-10 ARTICLES The Challenge and Promise of Blastocyst Cryopreservation by Marius Meintjes, PhD Presbyterian/Harris Methodist ARTS Program, 8160 Walnut Hill Lane, Perot Building, Suite 116, Dallas, TX 75231 Introduction Blastocyst culture and transfer has been promoted as a novel approach to reduce multiple pregnancies. With the high implantation rates and the resultant high percentage of twin pregnancies observed when transferring fresh blastocysts, single blastocyst transfers should be considered. With a reliable blastocyst cryopreservation program in place providing consistent acceptable frozenthawed blastocyst transfer outcomes, single blastocyst transfer should be a logical next step to curtail the current epidemic of twin pregnancies experienced with assisted reproduction. However, the frequent experience of less than attractive success rates with frozen-thawed blastocysts discourages many centers, as cumulative fresh-frozen pregnancy rates may seem unacceptable. (Graph 1). Patients with only poor-quality ICM’s at the time of transfer may expect a high rate of biochemical pregnancies. However, ongoing pregnancies and live births are possible. Several healthy postthaw trophoblasts with no discernable ICM (ICM-grade F, Figure 2) were MARIUS MEINTJES, PHD transferred, which Post-thaw inner cell mass (ICM) survival resulted in clinical pregnancies followed by firstA detailed blastocyst-specific grading system has been trimester miscarriages. Similarly, weak and/or damaged developed over the years in our program for fresh and ICM's in the poor-quality ICM group may explain the frozen blastocyst evaluation (Figure 1). High biochemical high rate of biochemical pregnancies and the clinical pregnancy losses observed. to improve blastocyst It is tempting to speculate that assisted Efforts cryopreservation should focus to hatching may improve frozen-thawed a significant degree on ICM blastocyst transfer outcomes. survival. ICM survival may be improved by proper selection of pregnancy rates and miscarriage rates are frequent blastocysts for cryopreservation, pre-freeze cytoskeletal observations when transferring frozen-thawed stabilization, optimisation of cryoprotectant solutions, or blastocysts. A biochemical pregnancy is defined as a the application of ultra-rapid vitrification procedures. positive ßhCG result failing to progress to a clinical pregnancy. It has been observed that ICM’s are A place for assisted hatching of frozen-thawed preferentially damaged by cryopreservation and that the blastocysts? observed clinical outcomes directly relate to ICM quality It is documented that the composition and function of the zona pellucida may change due to freezing and thawing. It is tempting to speculate that assisted hatching may improve frozen-thawed blastocyst transfer outcomes. In a randomized prospective study comparing assistedhatched (n=76) and non-hatched (n=76) frozen-thawed blastocyst transfer cycles, it was observed that the implantation rate was improved with assisted hatching. However, there was no effect on the ongoing pregnancy/live birth rate (Graph 2). CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 12 • VOLUME 4 • FERTILITY MAGAZINE 11 ARTICLES day 6 (n=301 patients – Graph 3). The male:female sex ratio (57:43) of babies born from blastocysts frozen on day 5 was significantly skewed and similar to that reported for fresh blastocyst transfers. Interestingly, the male:female sex ratio (54:46) was not different for blastocysts frozen on day 6. C ONTINUED FROM PAGE 11 Day-5, day-6 cryopreserved blastocysts and sex ratio The exact timing and blastocyst developmental stage at the time of cryopreservation may be imperative to optimal frozen-thawed blastocyst pregnancy outcomes. Embryologists frequently face decisions in freezing expanding blastocysts with reduced cell numbers on day 5 or freezing the next day with increased cell numbers, but risking ICM degeneration or in-vitro hatching. It has been documented that fresh blastocyst transfers may result in an increased male:female sex ratio. Freezing the slower blastocysts (not selected for fresh transfer) may possibly result in a normalization of the sex ratio in frozen-thawed blastocyst pregnancies. It was observed that blastocysts frozen late on day 5 resulted in higher implantation and ongoing pregnancy/live birth rates than blastocysts frozen early on 12 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG Expectations from an optimized blastocyst cryopreservation program Even with the limitations of slow-rate blastocyst freezing, live birth rates close to that of fresh blastocyst transfers can be achieved. The optimization of a blastocyst cryopreservation program mandates a comprehensive approach. A sequential embryo culture system with attention to the protein supplement and a low oxygen environment has been shown to increase the number of blastocysts available for cryopreservation, improve the ICM quality and blastocyst post-thaw survival. Critical timing of cryopreservation seems essential as the success of slow-rate blastocyst cryopreservation appears to be developmental stage specific. Freezing and thawing protocols/solutions are crucial, however, only a part of the overall cryopreservation scheme. Table 1 summarizes agespecific results that can be expected with slow-rate cryopreservation and thawing of blastocysts when attention is given to the factors mentioned. Interestingly, it seems that as long as quality blastocysts are frozen, pregnancy rates are not significantly affected by the age of the patient. Patient age Number of Blastocysts/ Implantations Ongoing/ (years) transfers Transfer (%) live births (%) ≤34 94 1.7 45.1 54 35 to 37 41 1.7 39.4 42 38 to 40 25 1.7 53.5 60 >40 6 1.7 50 67 Donor 47 1.9 40.9 60 oocytes Total 213 1.8 44.2 54 T able 1. Ongoing pregnancy/Live birth outcomes for agespecific frozen-thawed blastocyst transfers Conclusions It appears that the success of blastocyst cryopreservation and thawing depends on the ability to protect and recover a viable ICM (Figure 3). Assisted hatching seems to be of little value if optimum culture conditions are established in the laboratory. Freezing of fully expanded, but not hatched, blastocysts before 118 hours post insemination results in the best clinical outcomes. Frozen-thawed blastocyst live birth rates equivalent to or better than those obtained with fresh blastocysts for patients of all ages should now enable IVF programs to offer routine fresh single-blastocyst transfers. Experience by embryologists, ARTICLES not only with the freezing and thawing procedure, but also with selecting appropriate blastocysts for cryopreservation, is essential for a quality blastocyst cryopreservation program. However, biochemical and miscarriage rates are still significantly higher than those observed for fresh blastocyst transfers. Even though blastocyst cryopreservation can be implemented successfully, refinements to current protocols or investigation of alternative techniques will be necessary to further improve ICM survival after thawing. Acknowlegements The author wishes to recognize Drs Brian Barnett, David Bookout, Samuel Chantilis, James Douglas, Rheza Guerami, Robert Kaufmann, Karin Lee, James Madden and Alfred Rodriguez as well as embryologists Hannalie Adriaanse, Tonya Davidson, Annelize De Kock, Oscar Perez, Mario Smuts and David Ward as the real reason for a successful blastocyst cryopreservation program. You can contact Marius Meintjes, PhD at: [email protected] Figure 2. An example of a hatching blastocyst 14 hours post thaw with a poor (F-grade) ICM and a “B” trophoblast grade. This embryo resulted in a biochemical pregnancy. Figure 1. ICM and trophoblast grading scores used to select blastocysts for cryopreservation and to identify good quality- and poor quality blastocysts before transfer. Figure 3. A hatching blastocyst 17 hours after thawing with a “B” ICM and an “A” trophoblast grade. This blastocyst resulted in a live birth. WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 13 14 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG ARTICLES Use of the Sperm Chromatin Structure Assay (SCSA ®) as a Diagnostic Tool in the Human Infertility Clinic by Donald P. Evenson, PhD, HCLD and Regina L. Wixon, PhD SCSA Diagnostics, Inc., Brookings Research & Technology Center, 807 32nd Avenue, Brookings, SD 57006, USA I nfertility has been attributed equally to men and women with 20% of infertility problems due to unknown factors. However, men’s infertility is rapidly surpassing women’s, according to infertility experts at the European Society for Human Reproduction and Embryology Meeting in Copenhagen, 2005. The decline in male fertility can be attributed to a number of factors, including but not limited to, environmental toxins, medications, infections, diet, drug use, high fever, elevated testicular temperature, binge drinking, air pollution, cigarette smoking and advanced age. What perhaps is an underlying effect of these insults on the fertility of men is an increase in sperm DNA fragmentation. The sperm chromatin structure assay (SCSA® ), (Evenson et al. 1980, (Palus et al. 2005), chemotherapy (Evenson et al. 1984a, Evenson et al. 1984b), and a variety of other chemicals that target different testicular germ cell types. Elevated sperm DNA fragmentation as measured by the SCSA has been shown to have a significant REGINA L. WIXON, PHD negative effect on pregnancy outcome. The % DONALD P. EVENSON, P HD, HCLD D N A below 30%. Data from this study Fragmentation Index (DFI) threshold were used to establish the statistical for infertility was first established in the comprehensive “Georgetown thresholds for the SCSA analysis of Male Factor Infertility Study” >30% DFI for ‘significant lack of’, >15-<30% DFI for ‘reasonable’ and <15% DFI for ‘high’ fertility potential status. To evaluate the effectiveness of the SCSA analysis, meta-analyses were conducted on studies of intercourse, IUI, routine IVF and ICSI methods of fertilization. A (Evenson et al. 1999). The study meta-analysis of three studies included 144 couples trying to (n=1575) was conducted to conceive naturally. SCSA data from male partners of 73 couples that investigate the relationship of sperm DNA fragmentation on pregnancy achieved pregnancy during months outcome using intercourse and IUI. 1-3 (Group 1) were used as the The meta-analysis indicated that standard of sperm DNA integrity compatible with high fertility. Group patients were 7.3 times more likely to achieve a pregnancy/delivery if the 1 had significantly different DFI DFI was < 30% (p=0.0001) (Evenson values compared to 40 couples et al. 1999, Spano et al. 2000, Bungum achieving pregnancy in months 4-12 (Group 3, P<0.01) and 31 couples not et al. 2004). When routine IVF alone was considered, couples were 2.2 achieving pregnancy (Group 4, P<0.001). The couples in this study were 6.5 times more likely to achieve CONTINUED ON PAGE 16 a pregnancy when their DFI was The application of PGD is increasing on an almost daily basis. Evenson et al. 1985) now in use for over two decades, has proved to be a very sensitive and precise measure of sperm DNA damage resulting from environmental toxicants or other damaging agents. Agents studied have been radiation (Sailer et al. 1995), heat (Sailer et al. 1997), air pollution (Selevan et al. 2000), smoking (Potts et al. 1999), insecticides (Xia et al. 2005, Bian et al. 2004), PCBs and persistent organochlorine pollutants (RignellHydbom et al. 2004), industrial chemicals (Sanchez-Pena et al. 2004), phthalates (Duty et al. 2003), arsenic WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 15 ARTICLES C ONTINUED FROM PAGE 15 times more likely to become pregnant if their DFI was <30% (n=521, p=0.0008) (Bungum et al. 2004, Henkel et al. 2004, Larson-Cook et al. 2003, Adams et al. 2004). A metaanalysis of five studies using ICSI and/or IVF (n=216) showed a nonsignificant trend where patients were 1.7 times more likely to achieve a pregnancy/delivery if the %DFI was <30% (p=0.11) (Bungum et al. 2004, Larson-Cook et al. 2003, Chohan et al. 2004, Morris et al. 2002, Larson et al. 2000). These meta-analyses show that the SCSA infertility test is significantly predictive for reduced pregnancy success using intercourse, IUI and routine IVF and, to a lesser extent, ICSI. While environmental insults are a concern for men’s infertility, the natural aging process is also associated with increasing levels of sperm DNA fragmentation. The latest research indicate that while some older men are highly fertile, the age of 47 years intersects with the 30% statistical threshold associated with a negative pregnancy outcome (see Figure 1). As seen in Figure 1, for the factor of sperm DNA integrity alone, a 50 year old man has on the average, a 30% decreased chance for a successful pregnancy relative to a man in his 20’s. Morris et al. (2002), also found sperm DNA damage to be strongly associated with age in men 29-44 years (r=0.669, p=0.032). Infertile couples less than 35 years of age (for both male and female) had a 53% pregnancy rate in comparison to 4% in infertile couples greater than 40 years of age (Klonoff-Cohen and Natarajan 2004). Since a quarter of men entering a fertility clinic are over the age of 40, aging and men’s fertility become a significant factor. When sperm DNA fragmentation is elevated, a trend for spontaneous miscarriages has been 16 FERTILITY MAGAZINE • VOLUME 4 • reported in several studies. This was first found in the “Georgetown Male Factor Infertility Study” where the DFI statistical groups were first identified. This study predicted almost half of the miscarriages in couples that were using intercourse and had elevated sperm DNA fragmentation values (Evenson et al. 1999). In two other studies, IVF/ICSI couples with elevated DFI values also found a trend in miscarriages (Virro et al. 2004, Check et al. 2005). In another study, twice as much sperm DNA fragmentation was found in couples with unexplained recurrent pregnancy loss in comparison to the general population (Carrell et al. 2003). Using a light microscope method of analysis, Morris et al. (2002) found that elevated sperm DNA damage was predictive of embryo development failure in a group of IVF/ICSI men. Three of the nine pregnancies (33%) in the elevated sperm DNA damaged group resulted in an early miscarriage while all six of the pregnancies in the low sperm DNA damaged group yielded live births. Who should have a sperm DNA fragmentation test? • All couples with unexplained infertility • Couples with a history of spontaneous miscarriages • Men over 40 years of age • Men with above listed pathologies • Men with exposure to toxic agents • Men with a questionable varicocele for repair Many patients with elevated sperm DNA fragmentation want to know how to “fix” their fragmentation. Treatment may include taking antioxidants, an improved healthy lifestyle, avoidance of hot tubs and not using a laptop computer on lap. Many couples have depleted financial resources and lost precious WWW.FERTILITYMAGAZINE.ORG time with ART procedures that were perhaps not appropriate for their infertility treatment. Physicians will typically use the SCSA as part of their patients’ initial infertility workup. The results of the SCSA analysis enables the physician to better manage their patients’ infertility care, knowing that when the sperm DNA fragmentation levels are consistently elevated, IUI should be skipped and the patient move onto routine IVF or preferably ICSI. This saves the patient emotional and financial costs. References Adams C, Anderson L, Wood S (2004) High, but not moderate, levels of sperm DNA fragmentation are predictive of poor outcome in egg donation cycles [abstract no. O-110]. In: Abstracts of the Scientific Oral & Poster Sessions. Philadelphia, PA: Journal of the American Society for Reproductive Medicine, (with permission):S44. Bian Q, Xu LC, Wang SL, Xia YK, Tan LF, Chen JF, Song L, Chang HC, Wang XR (2004) Study on the relation between occupational fenvalerate exposure and spermatozoa DNA damage of pesticide factory workers. Occup Environ Med, 61, 999-1005. Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A (2004) The predictive value of sperm chromatin structure assay (SCSA®) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod, 19, 1401-1408. Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, Campbell B (2005) Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl, 49, 49-55. Check JH, Graziano V, Cohen R, Krotec J, Check ML (2005) Effect of an abnormal sperm chromatin structural assay (SCSA ®) on pregnancy outcome following (IVF) with ICSI in previous IVF failures. Arch Androl, 51, 121-4. Chohan KR, Fiffin JT, Lafromboise M, DeJonge CJ, Carrell DT (2004) Sperm DNA damage relationship with ARTICLES embryo quality and pregnancy outcome in IVF patients [abstract no. O-137]. In: Abstracts of the Scientific Oral & Poster Sessions. Philadelphia, PA: Journal of the American Society for Reproductive Medicine, (with permission):S55. Duty SM, Singh NP, Silva MJ, Barr DB, Brock JW, Ryan L, Herrick RF, Christiani DC, Hauser R (2003) The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay. Environ Health Perspect, 111, 1164-9. Evenson DP, Darzynkiewicz Z, Melamed MR (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science, 210, 1131-3. Evenson DP, Higgins PJ, Grueneberg D, Ballachey BE (1985) Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry, 6, 23853. Evenson DP, Jost LK, Zinaman MJ, Clegg E, Purvis K, de Angelis P, et al (1999) Utility of the sperm chromatin structure assay (SCSA®) as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod, 14, 1039-1049. Evenson DP, Klein FA, Whitmore WF, Melamed MR (1984) Flow cytometric evaluation of sperm from patients with testicular carcinoma. J Urol, 132, 1220-5. Evenson DP, Arlin Z, Welt S, Claps ML, Melamed MR (1984) Male reproductive capacity may recover following drug treatment with the L10 protocol for acute lymphocytic leukemia. Cancer, 53, 30-6. Klonoff-Cohen HS, Natarajan L (2004) The effect of advancing paternal age on pregnancy and live birth rates in couples undergoing in vitro fertilization or gamete intrafallopian transfer. Am J Obstet Gynecol, 191, 507-14. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al (2004) Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril, 81, 965-972. Larson-Cook K, Brannian JD, Hansen KA, Kasperson K, Aamold ET, Evenson DP (2003) Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril, 80, 895-902. Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP (2000) Sperm Relationship between Age and DNA Fragmentation Figure 1. CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 18 • VOLUME 4 • FERTILITY MAGAZINE 17 ARTICLES C ONTINUED FROM PAGE 17 chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod, 15, 1717-1722. Morris ID, Ilott S, Dixon L, Brison DR (2002) The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod, 17, 990-8. Palus J, Lewinska D, Dziubaltowska E, Stepnik M, Beck J, Rydzynski K, Nilsson R (2005) DNA damage in leukocytes of workers occupationally exposed to arsenic in copper smelters. Environ Mol Mutagen, 2005 May 6; [Epub ahead of print] Potts RJ, Newbury CJ, Smith G, Notarianni LJ, Jefferies TM (1999) Sperm chromatin damage associated with male smoking. Mutat Res, 423, 18 FERTILITY MAGAZINE • VOLUME 4 • 103-11. Rignell-Hydbom A, Rylander L, Giwercman A, Jonsson BA, Lindh C, Eleuteri P, Rescia M, Leter G, Cordelli E, Spano M, Hagmar L (2005) Exposure to PCBs and p,p'-DDE and human sperm chromatin integrity. Environ Health Perspect, 113, 175-9. Spano M, Bonde J, Hjollund HI, Kolstatd HA, Cordelli E, Leter G (2000) Sperm chromatin damage impairs human fertility. Fertil Steril, 73, 43-50. Sailer BL, Jost LK, Erickson KR, Tajiran MA, Evenson DP (1995) Effects of Xirradiation on mouse testicular cells and sperm chromatin structure. Environ Mol Mutagen, 25, 23-30. Sailer BL, Sarkar LJ, Bjordahl JA, Jost LK, Evenson DP (1997) Effects of heat stress on mouse testicular cells and sperm chromatin structure. Androl, 18, 294-301. Sanchez-Pena LC, Reyes BE, LopezCarrillo L, Recio R, Moran-Martinez J, Cebrian ME, Quintanilla-Vega B (2004) Organophosphorous pesticide WWW.FERTILITYMAGAZINE.ORG exposure alters sperm chromatin structure in Mexican agricultural workers. Toxicol Appl Pharmacol, 196, 108-13. Selevan SG, Borkovec L, Slott VL, Zudova Z, Rubes J, Evenson DP, Perreault SD (2000) Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Environ Health Perspect, 108, 887-94. Virro MR, Larson-Cook KL, Evenson DP (2004) Sperm chromatin structure assay (SCSA®) related to blastocyst rate, pregnancy rate and spontaneous abortion in IVF and ICSI cycles. Fertil Steril, 8, 1289-1295. Xia Y, Cheng S, Bian Q, Xu L, Collins MD, Chang HC, Song L, Liu J, Wang S, Wang X (2005) Genotoxic effects on spermatozoa of carbaryl-exposed workers. Toxicol Sci, 85, 615-23. You can contact Donald P. Evenson, PhD and Regina L. Wixon, PhD at: [email protected] Air purification is as important to the IVF professional, as it is to the consumer. If it's good for an embryo, it’s good for you and your loved ones! Environmental air is receiving daily attention in the World News media as well as in all info-commercials. Coda® Air Purification System was developed and tested for IVF labs and embryo protection with continued reported success worldwide. It’s the only technology of its kind, scientifically designed and tested to provide the purest air required for an embryo development in IVF labs. Manufactured in clean room environment with the purest and tested filtration materials, by a medical ISO certified company with the strictest quality control system in place. Embryo & IVF Labs For the fertility professionals, the Coda® Aero has incorporated years of research and testing to purify the air in your IVF clinic, reproductive and research laboratory. The Aero has been proven to remove Air Contaminates, bacteria, mold and critically dangerous airborne pollutants. The Aero filters out all of these dangerous components and keeps them off of your specimen. Aero is manufactured under ISO 9002 and ISO 13485 worldwide regulations and guideline, CE registered and FDA compliant. The Consumer and IVF patient Consumers and Fertility patients benefit from the Coda® system developed for fertility clinics and proven to help improve embryonic development, pregnancies and childbirth. The Aero is consistent and proven to run for years to overcome demanding environments. The Aero is of commercial quality and has been proven to last. The Coda® Aero has a full 3 year warrantee and is dependable. These proven Aero is one of the few air purifiers that is FDA approved and produced in with Quality System, by an ISO:9002 company. • • • • Coda® Aero Filter system contains a 99.997% effective HEPA filter and pure Activated Carbon to remove all Volatile organic compounds, (VOCs), Chemical air contaminants (CAC), Bacteria, mold, fungus from your environment. The Areo is available for office and home use, is compact and light weight, equipped with a cost effective blower and is very economical to run. Order your Coda® Aero today and be sure that you are doing your best to purifying the air of your office personnel, family your children and loved ones. Protect yourself and others from harmful pollutants, environmental contaminants, dust and dirty air. Professional order today Consumers call Amy at customer service: IVFonline US/Canada: 800-720-6375 International: 519-826-5800 email: [email protected] Fax: 519-826-6947 519-829-5800 ext 200 Discount code #FW04 order Coda® Aero ordering ©2005 <genX> international US/Canada: 800-720-6375 International: 519-826-5800 Fax: 519-826-6947 email: [email protected] G-AeroCons-2005-10 ARTICLES The Effect of CO2 Concentration and pH on the In-Vitro Development of Mouse Embryos by Avner Hershlag, MD and Huai L. Feng, PhD, HCLD Center for Human Reproduction, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030,. U.S.A. Summary This study was designed to assess the effect of CO2 concentration on the pH of the medium and the developmental rates of mouse embryos. Pronuclear stage zygotes were placed in microdrops of HTF containing 10% SSS and cultured at 37°C, under 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5% CO2 in air for 120 h. The embryos were scored for cleavage at 24, 48, 72 and 96 hours and for blastocoel formation at 120hours of culture. Cleavage to the 2-cell stage was lower under 4.0% CO 2 (76.8%), but remained unchanged at all other CO 2 concentrations. Development to the blastocyst stage was 69.2% under 5.0% CO 2, and significantly lower at all other CO2 concentrations. The development of mouse embryos could be arrested in both high and low pH culture media. Introduction Exposure of gametes or embryos to potential toxins at any point during the IVF cycle may have a detrimental effect on embryogenesis and consequent pregnancy rates. Testing of factors such as CO2 concentrations of the incubator and pH of the culture medium is the backbone HUAI L. FENG, PHD AVNER HERSHLAG, MD microdrops and cultured under each of the CO 2 concentrations for 120 h (three microdrops per CO2 concentration). The embryos were scored for cleavage at 24, 48, 72 and 96 h, and for blastocoel formation at 120 h. The CO 2 concentration was Testing of factors such as CO2 concentrations of the measured using a CO2 analyzer (Mid Atlantic, Marlton, NJ), and pH incubator and pH of the culture medium is the backbone was determined with a digital pH of any stringent quality control program in IVF. microelectrode. of any stringent quality control program in IVF. Developmental rates of a one-cell (1) and two-cell (2) mouse embryo system have evolved into the standard bioassays for quality control in clinical IVF laboratories (3). In this study, one-cell mouse embryos were used to evaluate the effects of CO2 concentration and pH on invitro development. Materials and Methods Pronuclear stage zygotes (B6C3 F1 female/ B6D2F1 male mice) were obtained from Conception Technology Inc. (San Diego, CA). Microdrops (50µl) of HTF (LifeGlobal, Guilford, CT) supplemented with 10% SSS (Irvine, Santa Ana, CA), were set up and equilibrated at 37oC for 18 hours under 4.0%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0% or 7.5% CO2 in air. A total of 30-45 zygotes were then placed in the Results As shown in Figure 1, the pH of the media varied from 7.4 under 4.0% CO 2 to 6.8 under 7.5% CO2. Cleavage to the 2cell stage was lower under 4.0% CO2 (76.8%), but remained unchanged at all other CO2 concentrations. Further development to the blastocyst stage was 69.2% under 5.0% CO 2 and significantly less than that at all other CO2 concentrations (Figure 1). Discussion Carbon dioxide is required not only to maintain the pH of bicarbonate buffered medium, but is also readily incorporated into protein and nucleic acids by mouse embryo at all stages prior to implantation (4). Culture CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 22 • VOLUME 4 • FERTILITY MAGAZINE 21 ARTICLES C ONTINUED FROM PAGE 21 Figure 1. The effect of CO 2 concentration on the pH of the culture medium and development of mouse zygotes to the blastocyst stage. ** P<0.01 vs. 5% CO 2 (pH 7.2), * P< 0.05 vs. 5% CO2 (pH 7.2). Using Fisher’s Exact Test. systems for the preimplantation mammalian embryo routinely employ a carbon dioxide concentration of 5% coupled with a bicarbonate concentration of approximately 25 mM. However, the carbon dioxide concentrations of the uterine environment vary from 5% to 10% in different animal species (5). The optimal concentration of carbon dioxide for human embryo development has yet to be determined. According to the pH (7.1 to 8.0) of fluid collected from the reproductive tract of animal species, external pH of culture media formulated for preimplantation embryos is commonly between 7.3 ~7.4 (depending on the CO 2 concentration used) (5). Studies on the mouse embryos determined that development from the 2-cell stage to the blastocyst stage could occur in media with a pH range from 5.9 to 7.8 (6). The embryos at 8-cell stage from hamster could develop to the blastocyst stage in medium with an external pH range from 6.4 to 7.4 (7). Similarly in our studies, the mouse embryos from the zygote stage to the blastocyst stage could occur in HTF medium with a pH range from 6.8 to 7.4. However, the cleavage rate to the 2-cell stage was lower at pH 7.4 (76.8%), but remained unchanged at all other pH (6.8~7.3). Further development to the blastocyst stage was significantly decreased in both high pH 7.4 (at 4.0% CO2) and low pH 22 FERTILITY MAGAZINE • V OLUME 4 • WWW.FERTILITYMAGAZINE.ORG 6.8~7.0 (at 6.5-7.5% CO 2) culture environments. Development of mouse embryos could be arrested in both the high and low pH culture media. The prior reports indicated that even small fluctuations in pHi can significantly retard subsequent developmental competence. Fluctuations in either the acidic or the alkaline range can drastically reduce embryo development (5, 8, 9). Therefore, care should be taken to avoid fluctuations in the pH of media during gametes, embryo manipulation and culture. In most of laboratories, the use of an oil overlay not only reduces the harmful effects of increases in osmolarity, it also reduces changes in pH caused by a loss of CO2 from the media when culture dishes are taken out of the incubator for gamete or embryo examination. This is especially relevant for oocytes that are stripped of their cumulus before an ICSI procedure. These oocytes and embryos immediately following the procedure cannot regulate their ionic homeostasis, which may result in decrease fertilization rate and pregnancy rate. Conclusions The results show that proportion of of one-cell mouse embryos that develop to the blastocyst stage is reduced at both high and low pH. Optimal blastocyst development was observed at a 5.0% CO 2 concentration, which produced a pH of 7.2. References 1. Quinn P, Warnes , Kerin J.F. et al. Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fert Steril, 41, 202-9, 1984. 2. Ackerman S, Swanson RJ, Stokes G, and Veeck L., Culture of preimplantation mouse embryos as a quality control assay for human in vitro fertilization. Gamete Res, 9, 145-152, 1984. 3. McDowell JS, Swanson RJ, Maloney M, Veeck L., Mouse embryo quality control for toxicity determination in the Norfolk in vitro fertilization program. J In Vitro Fert Embryo Transfer, 5,144-148, 1988. 4. Graves C.N. and Biggers J.D., Carbon dioxide fixation by mouse embryos prior to implantation, Science, 167, 1506, 1970. 5. Trounson A.O. and Gardner D.K. Handbook of Ferilization, Second Edition, New York, CRC Press, 2000. 6. Ludwig T.E., Lane M. and Bavister B.D., Increased fetal development after transfer of hamster embryos cultured with glucose, Biol. Reprod., 58(suppl.1), 306, 1998. 7. Carney E.W. and Bavister B.D., Regulation of hamster embryo development in vitro by carbon dioxide, Biol. Reprod., 36, 1155, 1997. 8. John D.P. and Kiessling A .A., Improved pronuclear mouse embryo development over an extended pH range in Ham's F-10 medium without protein, Fertil. Steril., 49, 150-5, 1988. 9. Naaktgeboren N., Quality control of culture media for in vitro fertilization, Ann Biol Clin., 45, 368-72, 1987. You can contact Avner Hershalg, MD and Huai L. Feng, PhD, HCLD at: [email protected] ARTICLES Degree of Re-expansion of Thawed Blastocysts is Predictive of Frozen Embryo Transfer (FET) Success By Deborah C. Merryman, MSc, ELD/TS (ABB), Embryology Laboratory Director ART Fertility Program of Alabama, Birmingham, AL (Presented in part at the Annual Meeting of the American Society of Reproductive Medicine, Philadelphia, Pennsylvania, October 16-20, 2004, poster #P252, DC Merryman, SE Stringfellow, CA Yancey, VL Houserman, CA Long, and KL Honea.) INTRODUCTION Cryopreservation is an integral component of an in-vitro fertilization (IVF) program. Benefits from a successful cryopreservation program include cost effectiveness, enhancement of the cumulative pregnancy rate and reduction in the multiple birth rate with fresh IVF. A successful cryopreservation program is critical to those patients needing only a single blastocyst transferred with fresh IVF 1. Cryopreservation of excess embryos is routinely performed at the blastocyst stage at the ART Fertility Program of Alabama. Clinical pregnancy rates for cryopreserved embryos have been shown to be higher for the blastocyst as compared to other cell stages2. Some of the factors associated with frozen embryo transfer (FET) success with blastocysts include incubation time between thaw and transfer3 and the degree of blastocyst expansion at transfer4. OBJECTIVE Frozen-thawed blastocysts immediately contract after thawing and subsequently re-expand over time. The objective of this study was to evaluate the relationship between the re-expansion of frozen-thawed blastocysts two hours after thawing and pregnancy outcome. post thaw evaluation of frozen-thawed blastocysts was available. Blastocysts exhibiting at least a full blastocoel with a good inner cell mass and trophectoderm (grade 3BB, Gardner, et al 5), were frozen on day 5 or 6 of embryo culture. A two-step freeze and a two- or multi-step thaw procedure was DEBORAH C. MERRYMAN , MSC utilized with glycerol and sucrose in both the freeze and thaw solutions. After thawing, blastocysts were cultured in microdroplets of culture medium with 20 mg/ml protein under oil for 2 to 6 hours prior to transfer. Two hours after thawing, blastocysts were evaluated for degree of re-expansion. Re-expansion was assessed by observing the percentage viable/re-expanded per the total volume of the frozen blastocyst and documented in 25% increments (see figures). The replacement cycle routinely included estradiol, 2mg bid orally, for 10 to 14 days. Ideally, progesterone, 200mg vaginal capsules tid and 50 mg IM qd, was given when endometrium was 8mm or more and continued until pregnancy test. Blastocysts were thawed and transferred on day 6 of progesterone utilizing an echotip catheter (Cook) under ultrasound guidance. Fisher's exact test, Mann Whitney u-test or Student's t-test was used for statistical analysis as indicated. Statistical significance was defined as P <0.05. Cryopreservation is an integral component of an in-vitro fertilization (IVF) program DESIGN This study was designed to retrospectively review the outcome of FET cycles in relation to blastocyst reexpansion two hours post thaw. Outcome was evaluated by ongoing pregnancy and/or delivery rate (ODR), defined as the number of ongoing pregnancies and/or deliveries per embryo transfer, and implantation rate (IR), defined as the number of ongoing fetal heartbeats and/or live births per number of embryos transferred. Pregnancies and fetal heartbeats were considered ongoing at 12 or more weeks' gestation. RESULTS FETs were grouped according to the number of blastocysts transferred with 50% or more re-expansion MATERIAL AND METHODS The data consisted of 75 FET cycles in which a two-hour CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 24 • VOLUME 4 • FERTILITY MAGAZINE 23 ARTICLES C ONTINUED FROM PAGE 23 two hours after thawing: 0 (N=18) and 1 (N=57). A significant increase in ODR (0% vs. 33.3%, P=0.004) and IR (0% vs. 16.2%, P=0.02) was shown when one or more of the blastocysts transferred were 50% or more re-expanded two hours after thawing (Table 1). No ongoing and/or delivered pregnancies occurred when all blastocysts transferred were <50% re-expanded two hours after thawing. A significant increase in the total number of embryos transferred (1.7 v. 2.4, p=0.001) and the number of embryos transferred with 50% or more re-expansion two hours after thawing (0.0 v. 1.6, p<0.0001, Table 2) was seen when one or more of the blastocysts transferred were 50% or more re-expanded two hours after thawing. All other patient characteristics evaluated were not significantly different between groups. FETs were also grouped according to pregnancy outcome, specifically, cycles with an ongoing and/or delivered pregnancy (N=19) vs. cycles without an ongoing and/or delivered pregnancy (N=56). Characteristics showing a significant difference between groups included the total number of embryos transferred (2.6 v. 2.1, p=0.02), the number of embryos transferred with 50% or more reexpansion two hours after thawing (2.2 v. 1.0, p<0.0001), and the number of transfers with 1 blastocyst transferred at 50% or more re-expansion two hours after thawing (100% v. 67.9%, p=0.004, Table 3), with ongoing and/or delivered pregnancy vs. without ongoing and/or delivered pregnancy, respectively. from the transfer of three and one from the transfer of two re-expanded blastocysts and no ongoing or delivered triplet gestation within the data set. Of note, ongoing pregnancies and deliveries occurred from the thaw of blastocysts cryopreserved on day five, day six and from a combination of day five and six of embryo culture (data not shown). CONCLUSIONS 1. The assessment of the re-expansion of frozen-thawed blastocysts two hours post thaw is predictive of FET success. 2. The presence of one or more frozen-thawed blastocyst with 50% or more re-expansion two hours post thaw is related to FET success. On the other hand, the absence of one or more frozen-thawed blastocyst with 50% or more re-expansion two hours post thaw is related to FET failure. 3. IR and ODR can be optimized for FET cycles by evaluating the re-expansion of thawed blastocysts two hours post thaw. 4. Evaluation of the viability of frozen-thawed blastocysts can be performed two hours post thaw; extended incubation beyond two hours post thaw is not necessary. 5. Thawing of excess frozen blastocysts may be avoided by initially thawing the minimum number to be transferred followed by a two hour assessment and only then thawing more if needed. DISCUSSION For FET cycles, the ODR and IR increases with the number of re-expanded blastocysts transferred. While an ODR of 33.3% and an IR of 16.2% was achieved in FETs with one or more blastocyst transferred with 50% or more reexpansion (N=57), an ODR of 48.1% and an IR of 25.0% was achieved in FETs with two or more blastocysts transferred with 50% or more re-expansion (N=27) two hours after thawing. Of the 10 FETs with three, 17 FETs with two and 30 FETs with one re-expanded blastocyst transferred, there were three sets of twins (15.8%), two Table 1. Treatment outcome as a function of blastocyst re-expansion in a group of 75 FET cycles. Number of blastocysts transferred with ≥50% re-expansion 2 h post thaw 0 Frozen embryo transfers 24 ≥1 P 18 57 – Ongoing fetal heart beats and/or live births (IR) 0 (0) 22 (16.2) 0.02 Ongoing and/or delivered pregnancies (ODR) 0 (0) 19 (33.3) 0.004 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG ARTICLES Table 2. Summary of patient characteristics in relation to blastocyst re-expansion in a group of 375 FET cycles. Number of blastocysts transferred with ≥50% re-expansion 2 h post thaw 0 ≥1 P 18 57 – Age 33.7 ± 4.4 31.7 ± 4.8 NS Embryos transferred 1.7 ± 0.8 2.4 ± 0.8 0.001 Embryos transferred with 50% re-expansion 2 h post thaw 0.0 ± 0.0 1.6 ± 0.8 <0.0001 Embryos thawed 2.3 ± 1.7 2.7 ± 1.1 NS FSH, mIU/mL, highest basal or day 10 9.3 ± 1.6 9.5 ± 2.4 NS Body mass index 24.4 ± 5.5 26.2 ± 5.9 NS Endometrium, mm 9.4 ± 1.8 9.7 ± 1.3 NS Previous live birth 10 (55.6) 26 (45.6) NS 11b (20.4) NS Frozen embryo transfers a Previous live birth with IVF 5 (29.4) Values are average with standard deviation or number with percentage in parentheses. a 1 of 18 patients without previous fresh transfer attempt; b 3 of 57 patients without previous fresh transfer attempt. Table 3. Summary of patient characteristics in relation to pregnancy outcome in a group of 75 FET cycles. Pregnancy outcome Ongoing and/ or delivered Not Pregnant P 19 56 – FETs with 1 blastocyst transferred with 50% re-expansion 2 h post thaw 19 (100.0) 38 (67.9) 0.004 Age 32.5 ± 4.5 32.2 ± 4.9 NS Embryos transferred 2.6 ± 1.1 2.1 ± 0.7 0.02 Embryos transferred with 50% re-expansion two hours post thaw 2.2 ± 1.0 1.0 ± 0.8 <0.0001 Embryos thawed 3.1 ± 1.3 2.6 ± 1.3 NS FSH, mIU/mL, highest basal or day 10 10.3 ± 2.5 9.3 ± 2.2 NS Body mass index 25.9 ± 4.3 25.6 ± 6.3 NS Endometrium, mm 9.7 ± 1.4 9.6 ± 1.4 NS Previous live birth (%) 8 (42.1) 26 (46.4) NS 18b NS Frozen embryo transfers a Previous live birth with IVF (%) 6 (35.3) (33.3) Values are average with standard deviation or number with percentage in parentheses. a 2 of 19 patients without previous fresh transfer attempt; b 2 of 56 patients without previous fresh transfer attempt. 6. Multiple gestations can be reduced during FET cycles by limiting the number of blastocysts transferred with 50% or more re-expansion two hours post thaw. References 1. Tiitinen A, Halttunen M, Harkki P, et al. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001:1140-44. 2. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod BioMed Online 2003:36774. 3. Guerif F, Cadoret V, Poindron J, Lansac J, Royere D. 4. 5. Overnight incubation improves selection of frozen-thawed blastocysts for transfer: preliminary study using supernumerary embryos. Theriogenology 2003:1457-66. Behr B, Gebhardt , Lyon J, Milki A. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002:697-99. Gardner DK, Schoolcraft WB. In vitro culture of human blastocyst. In: Jansen R and Mortimer D, eds. Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. Carnforth, Parthenon Press, 1999:378-88. You can contact Debbie Merryman at: [email protected] WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 25 The Fertility Essentials to enhance your reproductive system NOW! Discover the Power of Lotus by fertileage TM Order Online at www.FertileAge.com US/Canada: 800-720-6375 • International: 519-826-5800 • Fax: 519-826-6947 • email: [email protected] ©2005 FertileAge FA-Essentials-2005-05 AGE & FERTILITY Advanced Maternal Age and Infertility by Alan B. Copperman, MD Director of Reproductive Endocrinology, Vice-Chairman of Obstetrics and Gynecology, Mount Sinai School of Medicine Co-Director, Reproductive Medicine Associates of New York, www.rmany.com A pervasive issue in the field of infertility today is advancing maternal age and its impact on conception. Though males experience some impairment in their fertility around age 45 plus, females begin to incur decline even prior to age 35. Because the world is moving towards social equality, women are waiting until a later age to start a family in order to mature and attain a stronghold in the workplace, and reproductive specialists are finding an increasingly larger population of women requesting treatment due to advanced age. Infertility is defined as the inability to conceive after a year of unprotected intercourse in women under 35 years of age, after six months in women 30 or older, or the inability to carry a pregnancy to term. Why does this age disparity exist between women under 35 and women over 35? Although 35 should not be considered as a absolute cut-off, many studies show that the chances of conceiving rapidly deteriorate, and the incidence of miscarriage increases. After age 40, the chances of a woman conceiving using her own eggs declines at an even greater rates. There are a number of interacting causalities associated with age advancement that engender difficulty in conception. Women are born into this world possessing a fixed egg pool – the number of eggs in the ovaries being about 2 million eggs. By adolescence, most of the eggs are depleted and only about 500,000 are left. With the onset of menstruation, follicle stimulating hormone (FSH) is released by the pituitary gland each month to stimulate egg maturation. A number of eggs will start to mature but, in general, only one egg will progress fully and be ovulated. Eventually, a woman will exhaust her ovarian reserve and will experience diminished ovarian reserve or even ovarian failure, and the production of eggs, estrogen and progesterone ceases. At this point, her only realistic chance of conception will be through egg donation, where she uses another woman’s eggs for conception and has the embryo implanted following in-vitro fertilization. exposure to radiation, and other external contaminants have been implicated in this decline, much is predetermined by genetics, leaving little for a patient to control. How to Test for Ovarian Reserve Quality Assessing the quality of ovarian reserve is ALAN B. COPPERMAN , MD the most effective way to test for how healthy and abundant the eggs are. Although there is no absolutely accurate and reliable test for ovarian reserve quality, a few exist that can provide the physician an initial direction of which treatment should be pursued. One such test is the Day 3 FSH test, is a simple blood test administered on the 3rd day of a woman’s menstrual cycle. A woman’s FSH level is the most reliable indicator of egg quality, because FSH is produced in greater quantities to stimulate ovarian growth as the eggs are depleted. Therefore, a high FSH suggests that the woman’s ovarian reserve is poor; while a low FSH suggests that the woman's eggs are healthy and she has a better prognosis for fertility. Estradiol (E2) levels can also indicate the health of the ovarian reserve, and most times is measured in conjunction with Day 3 FSH. Estradiol is a form of estrogen that also reaches elevated levels prior to ovarian failure in order to stimulate ovulation. Estradiol levels can be measured in the same blood sample that is taken for the measurement of FSH. The clomiphene citrate challenge test (CCCT) requires a slightly greater effort on the woman’s part. A woman taking this blood test initially has her baseline FSH levels drawn on day 3 through 5 of her menstrual With increasing age, women become more prone to infertility because of a decline in egg quality Age Factors Affecting Fertility With increasing age, women become more prone to infertility because of a decline in egg quality (often referred to as ovarian reserve). While smoking, pollution, CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 28 • VOLUME 4 • FERTILITY MAGAZINE 27 AGE & FERTILITY C ONTINUED FROM PAGE 27 cycle. She then takes Clomid for 5 days and has her FSH levels drawn again on the 10th day. FSH levels that have not reverted to normal baseline level by the 10th day indicate a poor ovarian reserve. The CCCT is a more effective test but not as commonly used due to the inconvenience of having to make multiple trips to the medical office. Basal antral follicle count is now considered a routine part of an ultrasound evaluation. By counting the number of immature follicles, the physician gets valuable information about the ovaries potential to produce eggs. Treatment Options Although a woman can undergo hormonal treatment such as clomiphene citrate therapy to overcome fertility complications due to advanced maternal age, the probability of conception will remain relatively low if she exhibits poor preliminary testing. In fact, in patients with early evidence of diminishing ovarian reserve, many fertility specialists advocate a rapid progression to IVF as the most effective treatment for these women. In IVF, a woman’s eggs and male’s sperm are retrieved and combined by an embryologist in an IVF laboratory. The embryos are examined as they grow for 3-5 days, and the 28 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG strongest and most viable embryos are implanted back into the woman’s uterus. Advancements in technology have increased success rates for IVF; but the chances for conception do decrease the older a woman gets. For women who suffer from ovarian failure, donor egg IVF offers a greater chance for fertility. In patients with contraindications to pregnancy or with a damaged or absent uterus, a gestational carrier (surrogate) can have a couple's embryo implanted and carry the baby to term. Conclusion The struggle between career and family is an ongoing struggle and, coupled with the financial pressures, many couples do not attempt to start a family until they are in their 30’s or even 40’s. Hundreds of thousands of years would have to pass before evolution could accommodate our changing human race, and medical technologies are advanced but there is a threshold of what it can do for conception. Though women should not feel pressured to procreate before they are financially and emotionally ready, but they need to be aware of the limitations in fertility and the impact of the biological clock on the reproductive system. You can contact Alan B. Copperman, MD at: [email protected] AGE & FERTILITY ART and Aging by Michael S. Neal, BSc, MSc, PhD candidate Centre for Reproductive Care, Hamilton Health Sciences, & Reproductive Biology Division, Department of Obstetrics & Gynecology, McMaster University, Hamilton, Ontario, Canada D emographic and epidemiological studies consistently show that infertility is closely related to female age. This has become apparent in our society as more women are delaying childbirth and turning to assisted reproductive technologies in an attempt to beat their reproductive biological clocks [1]. The basis of the correlation between age and infertility involves several factors. From a biological standpoint, the most important of these is egg quantity and quality. Oocyte quantity The ovary has been eloquently likened to an hourglass, with the female germ cells considered analogous to the grains of sand that trickle through the narrow opening depletion of these cells varies from person to person, with dramatic consequences on the fertility of women as they age. Poor response to ovarian stimulation during IVF treatment, is an indicator of diminishing ovarian reserve, which in turn has been shown to lead to reduced fecundity and earlier onset of menopause [4]. Since a woman’s endowment of oocytes is defined while she herself was an embryo, the influence of inutero exposure(s) can have a dramatic effect. As a result, recent concerns are focused on the impact of environmental contaminants on fetuses, as chemical insults in utero have been shown to result in impaired postnatal fertility [5]. Part of this effect may be due to reducing the initial pool of germ cells The basis of the correlation between age and infertility involves several factors. between the upper and lower chamber to account for the passage of time [2]. At birth, the finite number of female germ cells have already been established, although recent work published by Johnson et al [3] has challenged this dogma by showing that postnatal mouse ovaries possess mitotically active germ cells. However, this very small population of cells would not be able to turn around the effects of aging over the individual's lifespan. With each cycle the initial endowment provided at birth is depleted. Once the complete oocyte reserve has been exhausted, ovarian senescence occurs and women experience menopause. But, the size of follicle reserve at birth, and the rate of available at birth. Evidence to support this notion comes from a study showing that offspring from in-utero nicotine exposed rats had fewer follicles in the ovary, and significantly smaller litter sizes compared to saline controls [6]. Furthermore, constituents of cigarette smoke has been shown to be cytotoxic to developing germ cells [7]. Follicular Atresia Aside from the beginning number of follicles in the ovarian pool, the rate at which these follicles are recruited and released is critical in determining reproductive lifespan. Postnatal germ cell loss resulting from follicle atresia occurs MICHAEL S. NEAL, BS C, MS C, PHD CANDIDATE through a mechanism of programmed cell death called apoptosis. Oocyte loss occurs either directly from oocyte apoptosis or indirectly from apoptosis of the supporting (granulosa) cells that nourish and mature the oocyte through postnatal life. The rate at which the female biological clock ticks can be dramatically accelerated by apoptosis. For example, sterility and premature menopause are known to occur in young women following chemotherapy or radiation treatment for cancer [8]. A more subtle effect is seen in women who smoke. Female smokers take longer to conceive naturally [9], and have poorer pregnancy rates using IVF [10], are 2.5 time more likely to have diminished oocyte reserves [11, 12], and reach menopause 2-4 years earlier than their nonsmoking counterparts [13]. Taken together, this data strongly suggests that avoiding environmental tobacco smoke is one way to prevent WWW.FERTILITY MAGAZINE.ORG CONTINUED ON PAGE 30 • VOLUME 4 • FERTILITY MAGAZINE 29 AGE & FERTILITY oocytes from women of advancing age. C ONTINUED FROM PAGE 29 shortening one's reproductive lifespan. Although, the reasoning behind the occurrence and regulation of apoptosis in the germ line is poorly understood, there is great interest in unraveling this mystery. Understanding the cellular and molecular mechanisms that activate and execute programmed cell death in the ovary has great implications for the management of infertility and also, perhaps, of the aging process itself. Oocyte quality Even staving off follicular atresia will not solve the parallel problem of chromosomal damage in ageing eggs. It is well established that the considerable increase in anueploidy among embryos from older women contributes to reduced fertility by increasing both implantation failure and pregnancy loss [14]. Almost 80% of the eggs from women aged 40-45, undergoing IVF showed chromosome deficiencies that likely accounts for the higher rates of miscarriage and birth defects seen in these women [15]. Oocyte quality is further implicated by the fact that women who use the eggs of younger ovum donors have conception rates expected for the age of the donor and not that of the recipient. Successful reproduction hinges on oocyte quality, which is largely determined by the genetic composition of the oocyte. Many key components of the egg contribute to normal embryogenesis including cytoplasmic maturation, spindle formation, energy supply, pronuclear formation, oocyte activation, syngamy, early cleavage events and embryonic development. Disruption or alteration of one or several of these factors will lead to embryonic demise and are more likely to occur in 30 FERTILITY MAGAZINE • VOLUME 4 • Summary For many women of reproductive age, controlling fertility is the main concern and in contrast being able to conceive when desired is considered easy. However, putting off child bearing to an older age, when fertility is in decline, has brought home the reality, and placed high expectations on what can be achieved with assisted reproductive technologies. Even with the transfer of multiple embryos, there is a marked agerelated decline in female fertility [16]. An admirable goal of all medical caregivers should be increased awareness to the general public of the link between fertility and aging. References 1. Leridon H. Can assisted reproduction technology compensate for the natural decline in fertility with age? A model assessment. Hum Reprod 2004; 19: 1548-1553. 2. Morita Y, Tilly JL. Oocyte apoptosis: like sand through an hourglass. Dev Biol 1999; 213: 1-17. 3. Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 2004; 428: 145-150. 4. Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, Seed P. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003; 18: 527-533. 5. Vidaeff AC, Sever LE. In utero exposure to environmental estrogens and male reproductive health: a systematic review of biological and epidemiologic evidence. Reprod Toxicol 2005; 20: 5-20. 6. Pettric JJ, Neal MS, Foster WG, Holloway AC. In utero and lactational exposure to nicotine: ovarian effects. Annual FL Johnson Research Symposia, Dept of Obstetrics and Gynecology, McMaster University. 2005. 7. MacKenzie KM, Angevine DM. WWW.FERTILITYMAGAZINE.ORG 8. 9. 10. 11. 12. 13. 14. 15. 16. Infertility in mice exposed in utero to benzo(a)pyrene. Biol Reprod 1981; 24: 183-191. Familiari G, Caggiati A, Nottola SA, Ermini M, Di Benedetto MR, Motta PM. Ultrastructure of human ovarian primordial follicles after combination chemotherapy for Hodgkin's disease. Hum Reprod 1993; 8: 2080-2087. Baird DD, Wilcox AJ. Cigarette smoking associated with delayed conception. Jama 1985; 253: 29792983. Neal MS, Hughes EG, Holloway AC, Foster WG. Sidestream smoking is equally as damaging as mainstream smoking on IVF outcomes. Hum Reprod 2005; 20: 2531-2535. Sharara FI, Beatse SN, Leonardi MR, Navot D, Scott RT, Jr. Cigarette smoking accelerates the development of diminished ovarian reserve as evidenced by the clomiphene citrate challenge test. Fertil Steril 1994; 62: 257-262. El-Nemr A, Al-Shawaf T, Sabatini L, Wilson C, Lower AM, Grudzinskas JG. Effect of smoking on ovarian reserve and ovarian stimulation in invitro fertilization and embryo transfer. Hum Reprod 1998; 13: 21922198. Jick H, Porter J. Relation between smoking and age of natural menopause. Report from the Boston Collaborative Drug Surveillance Program, Boston University Medical Center. Lancet 1977; 1: 1354-1355. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995; 64: 382-391. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996; 11: 22172222. Templeton A, Morris JK, Parslow W. Factors that affect outcome of in-vitro fertilisation treatment. Lancet 1996; 348: 1402-1406. You can contact Michael S. Neal, BSc, MSc, PhD candidate at: [email protected] AGE & FERTILITY An Overview of the Effects of Age on Fertility in Women by Don Rieger, PhD LifeGlobal, LLC, Guelph, Ontario, Canada I. INTRODUCTION For a variety of social and economic reasons, women (and couples) are putting off having babies until they are in their mid-thirties, or later. As shown in Figure 1, this trend is common to most industrialized countries. In the United States, the average maternal age at first birth increased from 21.4 to 24.9 years of age from 1970 to 2000. Overall, birth rates in the United States decreased from 1970 to 1980, but since then, the birth rates for women over 30 years of age have increased significantly (Figure 2). Figure 1. The average maternal age at first birth in 1970 and 2000 for ten industrialized countries. (Mathews and Hamilton 2002) infertile is approximately twice as great for women 40-44, compared with women 30-34. It is important to note that the onset of agerelated infertility o c c u r s approximately 10 years before menopause (te Velde and Pearson, 2002). The decrease of fertility with age, DON RIEGER , PHD coupled with the tendency toward later child-bearing has led to the suggestion that “female ageing … is now the main limiting factor in the treatment of infertility” (Ford et al. 2000). Figure 3. The effect of a woman's age on infertility and the chance of remaining childless. Adapted from Menken et al. (1986). Figure 2. The birth rate for women 30-34, 35-39 and 40-44 years of age in the United States from 1970 to 2000. (U.S. National Center for Health Statistics 2003) * Historical data based upon the age at which a woman marries. This paper is intended as an overview of the information available on the relationships between age and fertility in women. It is by no means an extensive review of the literature, but rather, the data have been selected to illustrate general principles. Moreover, it is becoming increasingly evident that the fertility of a couple decreases with age of the male, as has been recently reviewed by Fisch (2005). The effect of male age is beyond Although some women can, and do, have babies into their forties and even fifties, in general, fertility decreases markedly with a woman's age, particularly from 35 years onward. As shown in Figure 3, the chance of being CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 32 • VOLUME 4 • FERTILITY MAGAZINE 31 AGE & FERTILITY C ONTINUED FROM PAGE 31 the scope of this paper, but where relevant and whenever possible, studies have been chosen that have been controlled or adjust ed for male age as well as frequency of intercourse and lifestyle factors. II. THE EFFECT OF AGE ON NATURAL CONCEPTION The birth of a normal healthy baby requires that a woman be able to ovulate a mature, normal oocyte (egg) at the appropriate time, that a fertile sperm be present within the oviduct (Fallopian tube) for fertilization, that the oviduct and uterus be capable of supporting the development of the embryo, and that the embryo (later the fetus) reside in uterus until fully developed and delivered without major complications. Many of these processes have been shown to be affected by the woman's age. The development of the oocytes and the follicles in which they reside begins when the woman is herself a fetus. Primordial germ cells develop into oogonia which divide and differentiate into primary oocytes. The primary oocytes are enclosed in a single layer of granulosa cells to form primordial follicles, numbering approximately 7,000,000 in both ovaries at 4-5 months of gestation. From there onward, the oogonia stop dividing, and the primary oocytes are arrested in development until puberty. Most of the primordial follicles are lost to atresia such that only approximately 1,000,000 are left at birth, 40,000 at the time of puberty and 1,000 at menopause. Throughout reproductive life, groups of the primordial follicles spontaneously grow and develop into early antral follicles. Most of these are also lost, except for (usually) one follicle per menstrual cycle that continues to develop into a large antral (dominant) follicle in response to gonadotrophic hormones (FSH and LH) secreted by the anterior pituitary gland. A surge of LH at mid-cycle induces the final maturation of the oocyte in the dominant follicle and its release (ovulation) into the oviduct where it can be fertilized. (See Piñón, 2002) The ability to produce and ovulate that one oocyte is directly related to the number of antral follicles on the ovaries at that time. As shown in Figure 4, the number of antral follicles present on the ovaries declines with age. In addition to the significant decline in the number of antral follicles, increasing age is also associated with chromosomal and functional aberrations in the oocytes. Most notably, as shown in Figure 5, the frequency of aneuploidy (abnormal numbers of chromosomes) in human oocytes increases exponentially after 35 years of age. Although the sperm can also contribute to chromosomal defects in the embryo, defects in the aged 32 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG oocyte are thought to be the major cause of Downs Syndrome and other chromosomal abnormalities in newborns (Figure 6). Increased maternal age is also associated defects in oocyte mitochondria, a structure responsible for energy production and many other important functions. These include an increased rate of point mutations in oocyte mitochondrial DNA (Barritt et al. 2000), and with a decreased ability of the mitochondria to produce energy (Wilding et al. 2002). Figure 4. The mean number of antral follicles detected by ovarian ultrasound in women. The values are derived from the regression lines presented in Scheffer et al. (1999). Figure 5. The effect of a woman's age on the frequency of aneuploidy in human oocytes (Pellestor 2004). Figure 6. The effect of maternal age on the risk of Downs Syndrome and other chromosomal abnormalities in newborns (Simpson and Elias 1994). AGE & FERTILITY As discussed in the following section on assisted reproduction, there is evidence that maternal age can have effects on fertilization and on development of the embryo. In natural conception, it is difficult to be certain whether problems of fertility arise from deficiencies in the embryo or deficiencies in the reproductive tract. However, it is very clear that the ability to get pregnant, to maintain the pregnancy, and to deliver a healthy baby decreases with maternal age. Figure 7 shows that the probability of establishing a clinical pregnancy following intercourse on the most fertile day of the cycle decreases steadily with increasing maternal age. Even for women who do eventually become pregnant, the likelihood of achieving conception within six months is dramatically reduced for women 35 years of wide variety of perinatal complications in the mother and the baby are significantly greater in women older than 35 years of age compared with women from 18-34 years of age (Figure 11). For example, compared with women 1834 years old, the risk of an emergency Caesarian section was 1.5 times as great for women 35-40 years old and more than twice as great for women older than 40. Based on historic data and on animal studies, Tarin et al. (2005) have suggested that delayed motherhood may also have long-term effects on the health of the children, including impaired fertility and reduced lifespan. Figure 9. The effect of maternal age on the incidence of miscarriage (Gindoff and Jewelewicz 1986). age and older (Figure 8). Figure 7. The effect of a woman's age on the probability of establishing a clinical pregnancy by intercourse on the most fertile day of the cycle. The data have been adjusted for paternal age and controlled for frequency of intercourse and lifestyle factors (Dunson et al. 2002). Figure 10. The effect of maternal age on the frequency of preterm delivery (Astolfi and Zonta 1999). Figure 8. The effect of maternal age on the likelihood of achieving conception within six months (Ford et al. 2000). The data are adjusted for lifestyle factors and paternal age. When pregnancy is established, increasing maternal age has severely detrimental effects on the outcome. Many of these effects probably result from the effects of maternal age on the oocyte/embryo/fetus or the embryonic contribution to the placenta. Both the miscarriage rate (Figure 9) and the frequency of preterm delivery (Figure 10) increase with increased maternal age. The risks of a C ONTINUED ON PAGE 34 WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 33 AGE & FERTILITY CONTINUED FROM PAGE 33 Figure 11. The risk of perinatal complications for women 35-40 or >40 years of age compared with women 18-34 years of age (Jolly et al. 2000). significant effect on the clinical pregnancy rate following IUI, but for any given age of the man, the clinical pregnancy rate decreases markedly with increasing age of the woman. Similarly, the pregnancy rate resulting from IUI with sperm from fertile donors is also significantly reduced with increasing age of the woman (Figure 13). Figure 12. The effect of the woman's age on clinical pregnancy rate following intrauterine insemination with the partner's sperm (Brzechffa et al. 1998). III. THE EFFECT OF AGE ON THE SUCCESS OF ASSISTED REPRODUCTION Infertility can result from lack of ovulation, poor quality oocytes, blocked oviducts (Fallopian tubes), impotence in the man, inadequate sperm numbers, poor quality sperm, or a poor interaction between the sperm and the cervical mucus. Many of these problems (and infertility due to unknown causes) can be treated, or at least circumvented, by assisted reproductive technologies (ART). However, although ART procedures can improve the chances of a having a baby, the success rate decreases markedly with increasing age of the woman. Figure 13. The effect of maternal age on clinical pregnancy rate following intrauterine insemination with frozen donor sperm (Ferrara et al. 2002). 1. Intrauterine Insemination and Donor Insemination Intrauterine insemination (IUI) is the simplest form of ART. Semen is collected from the male partner by masturbation and then the sperm are usually washed to remove dead cells and other possible deleterious components of the seminal plasma. The washed sperm are then placed directly into the uterus via a catheter which has been passed through the cervix. This serves to avoid any problems of passage of sperm through the cervix or cervical mucus, and provides a greater number of sperm within the uterus to increase the chances of fertilization. Intrauterine insemination is also commonly used in conjunction with ovulation induction, in order to ensure optimal timing of insemination. Figure 12 shows that the age of the man can have a 34 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG 2. In-Vitro Fertilization In-vitro fertilization (IVF) was originally developed to overcome the problem of blocked oviducts but is now also used to treat male-factor infertility (low numbers or quality of sperm) and infertility for which there is no apparent cause. In general, the woman is treated with gonadotrophins to increase the number of antral follicles that fully develop. It is important to note that gonadotrophin treatment has no effect on the numbers of AGE & FERTILITY primordial follicles that develop to the antral stage – it only acts to rescue the follicles that have already developed to the antral stage and would normally be lost to atresia. When the follicles have reached the appropriate size, the woman is given human chorionic gonadotrophin to mimic the normal ovulatory LH surge, and induce final oocyte maturation. A needle is used to recover the oocytes from the mature antral follicles. For standard IVF, the oocytes are placed together with sperm from the partner or a donor and the sperm penetrate the oocyte naturally. In cases where only small numbers or immotile sperm are available, fertilization can be achieved by injection of a single sperm into each oocyte (ICSI). After fertilization, the resultant embryos are cultured for 2 to 6 days and then transferred back into the uterus of the woman. As shown in Figure 4, the number of antral follicles present on a woman's ovaries decreases with age and this results in a decreased number of oocytes that can be retrieved for following gonadotrophin treatment for IVF (Figure 14a). Moreover, the quality of the oocytes also decreases with increasing age (Figure 14b), resulting in a decreasing proportion of the oocytes that can be successfully fertilized in vitro (Figure 14c). none were suitable for transfer) more than doubled for women 41-42 years old compared with women younger than 35. This reflects the decrease in the numbers and quality of oocytes retrieved with increasing age. For cycles in which embryos were produced, the proportion of those embryos that developed to the fetal stage (the implantation rate) decreased from 28% in women less than 35 years old to only 8% in women 41-42 (Figure 16b). Of the embryos that did implant, 38% were subsequently lost (miscarried) in women 41-42 years old, compared with only 14% in women younger than 35 (Figure 16b). Figure 15. The effect of a woman’s age on the development of fertilized oocytes to the blastocyst stage by Day 5 of culture (Wiemer et al. 2002). Figure 14. The effect of a woman's age on a) the number of oocytes retrieved, b) chromatid separation rate of the oocytes, and c) in-vitro fertilization rate (Lim and Tsakok 1997). Embryo development in culture is also affected by the age of the woman. In the example shown in Figure 15, the proportion of fertilized oocytes that developed to the blastocyst stage by Day 5 was significantly reduced with increasing age of the woman. The effects of age of the woman on the outcome of ART using her own oocytes are strikingly demonstrated by the statistics for ART procedures for 2002 reported to the U.S. Centers for Disease Control (U.S. Department of Health and Human Services – Centers for Disease Control and Prevention 2004) that are shown in Figure 16. The data include a total of 81,888 treatment cycles which resulted in approximately 24,100 live births. First, as shown in Figure 16a, the cancellation rate (treatment cycles in which no embryos were created or Overall the reduced implantation rate and increased fetal loss rate in older women resulted in only 5% of embryos transferred developing to a live baby in women 41-42 years old compared with 23% in women younger than 35 (Figure 16d). For women 41-42 years old, only 11% of cycles started yielded a live birth compared with 37% for women younger than 35 (Figure 16e). This would mean that on average, a woman 41-42 years old would need 12 IVF treatment cycles to have a 75% chance of one live birth, compared with only 3 treatment cycles for a woman younger than 35. In addition to the decrease in live-birth rate with increasing age, pregnancies and babies resulting from ART in older women using their own oocytes are subject to the same problems of pre-term delivery, perinatal complications and chromosomal abnormalities seen with natural conception. 3. The Use of Donor Oocytes In cases where a woman has no ovaries or is otherwise unable to produce her own viable oocytes, oocytes may be obtained from other women. The donors are most often anonymous fertile, young women but may be a relative or C ONTINUED ON PAGE 36 WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 35 AGE & FERTILITY C ONTINUED FROM PAGE 35 friend of the patient. The donor is treated with gonadotrophins and oocytes collected as described in the preceding section. Sperm from the patient's male partner is usually used for fertilization and the resulting embryos cultured and then transferred into the patient. Interestingly, it appears that the patient's age has no appreciable effect on the ability to support a pregnancy. The live-birth rate for women receiving embryos created from donor oocytes is approximately 50% at all ages from 25 to 45 (U.S. Department of Health and Human Services – Centers for Disease Control and Prevention 2004). Of course, the babies born from donated oocytes have no direct genetic relationship to the patient. Figure 16. The effect of a woman's age on the a) cancellation rate, b) implantation rate, c) fetal loss, d) approximate babies born per embryo transferred, and e) live birth rate with in-vitro fertilization of non-donor oocytes in the United States in 2002. (Taken or derived from: U.S. Department of Health and Human Services – Centers for Disease Control and Prevention 2004). Clearly, common ART procedures can improve the chances of pregnancy but cannot overcome the deleterious effects of aging on numbers and quality of the oocytes. Based on a computer model, Leridon (2004) has calculated that ART can make up for only half of the births lost by postponing an attempt to become pregnant from 30 to 35 years, and less than 30% of the births lost by postponing from 35 to 40 years. Based on a literature review and their own data, Broekmans and Klinkert (2004) conclude that the prognosis for a successful pregnancy with IUI or IVF for women 44 or older “is flat zero.” There are, however, two specialized ART procedures, pre-implantation genetic diagnosis and oocyte cryopreservation, that can, or have the potential to, circumvent the effects of aging on fertility. IV. APPROACHES TO CIRCUMVENTING EFFECT OF AGE ON FERTILITY THE 1. Pre-Implantation Genetic Diagnosis As noted above, the frequency of chromosomal abnormalities in oocytes increases with age in women, and this results in increased frequencies of chromosomal abnormalities in the embryos, fetuses, and babies born. An early approach to this was to obtain cells from the fetus by amniocentesis or chorionic villus sampling for evaluation of the chromosomes. Fetuses with abnormal numbers of chromosomes were then aborted, in order to prevent the birth of chromosomally abnormal babies. More recently, it has become possible to determine the chromosome status of early embryos produced by ART, before they are transferred back into the patient (preimplantation genetic diagnosis, PGD). In this case, only embryos with normal chromosome numbers are transferred. A positive side effect of embryo selection following PGD is that the implantation and birth rates are increased because chromosomally abnormal embryos are often also developmentally compromised. An example is shown in Figure 17, where the implantation rate for embryos that had been tested and judged to chromosomally normal was 17.6% compared with 10.6% for embryos that had not been tested (and presumed to be a mixture of normal and 36 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG AGE & FERTILITY abnormal embryos). Pre-implantation genetic diagnosis is usually used for couples with some history of chromosomal or other genetic defects, recurrent miscarriage, or in older women. Based on the improved rates of development following PGD, it has been suggested that all embryos should be tested. Figure 17. Implantation rates for unselected embryos and for embryos judged as chromosomally normal by pre-implantation genetic diagnosis (Munné et al. 2003). so, but it is imperative that women be aware that fertility decreases significantly with age, particularly after 35 years of age. From a purely biological perspective, the best approach to ensuring fertility is for women to have their babies before they have reached their mid-thirties, but for many women, this is not a desirable or even practical option. At any given age, assisted reproduction techniques may improve the chances of becoming pregnant, but cannot make up for the loss of fertility due to the effects of aging on the numbers and quality of oocytes. References 2. Oocyte Cryopreservation When living tissues are deep-frozen (cryopreserved) under the appropriate conditions, all biological processes are arrested and aging of the tissue stops until it is thawed. This approach has been long used for the storage of sperm and embryos, and has recently been extended to oocytes. A major interest in oocyte cryopreservation is to preserve the possibility of fertility for young women that are due to undergo radiotherapy and chemotherapy for the treatment of cancer. Such treatments can have severely deleterious effects on the oocytes. By removing and freezing the oocytes, they are not exposed to the cancer treatments. After the patient has recovered from the cancer treatments and wants to start a family, the oocytes can be thawed and fertilized, and the embryos transferred back into her uterus. In the same way that cryopreservation can protect oocytes from cancer treatments, it could also be used to protect oocytes from natural loss and degeneration due to aging. Stachecki and Cohen (2004) have suggested that this may offer an approach to preserving fertility for women wishing to delay reproduction. Oocytes would be collected from young women and then cryopreserved until they are ready to begin their families. Although as yet largely experimental, the pregnancy rates from cryopreserved oocytes are improving. V. CONCLUSIONS There is a tendency for women in industrialized countries to delay having babies until their mid-thirties or later. There are important social and economic reasons for doing Astolfi P, Zonta LA (1999) Risks of preterm delivery and association with maternal age, birth order, and fetal gender. Hum Reprod 14, 2891-4. Barritt JA, Cohen J, Brenner CA (2000) Mitochondrial DNA point mutation in human oocytes is associated with maternal age. Reprod Biomed Online 1, 96-100. Broekmans FJ, Klinkert ER (2004) Female age in ART: when to stop? Gynecol Obstet Invest 58, 225-34. Brzechffa PR, Daneshmand S, Buyalos RP (1998) Sequential clomiphene citrate and human menopausal gonadotrophin with intrauterine insemination: the effect of patient age on clinical outcome. Hum Reprod 13, 2110-4. Dunson DB, Colombo B, Baird DD (2002) Changes with age in the level and duration of fertility in the menstrual cycle. Hum Reprod 17, 1399-403. Ferrara I, Balet R, Grudzinskas JG (2002) Intrauterine insemination with frozen donor sperm. Pregnancy outcome in relation to age and ovarian stimulation regime. Hum Reprod 17, 2320-4. Fisch H (2005) ‘The Male Biological Clock.’ (Free Press: New York) Ford WC, North K, Taylor H, Farrow A, Hull MG, Golding J (2000) Increasing paternal age is associated with delayed conception in a large population of fertile couples: evidence for declining fecundity in older men. The ALSPAC Study Team (Avon Longitudinal Study of Pregnancy and Childhood). Hum Reprod 15, 1703-8. Gindoff PR, Jewelewicz R (1986) Reproductive potential in the older woman. Fertil Steril 46, 989-1001. Hassan MA, Killick SR (2004) Negative lifestyle is associated with a significant reduction in fecundity. Fertil Steril 81, 38492. Jolly M, Sebire N, Harris J, Robinson S, Regan L (2000) The risks associated with pregnancy in women aged 35 years or older. Hum Reprod 15, 2433-7. Leridon H (2004) Can assisted reproduction technology compensate for the natural decline in fertility with age? A model assessment. Hum Reprod 19, 1548-53. Lim AS, Tsakok MF (1997) Age-related decline in fertility: a link to degenerative oocytes? Fertil Steril 68, 265-71. Mathews T, Hamilton B (2002) 'Mean age of mother, 1970-2000.' National Center for Health Statistics, Hyattsville, Maryland. CONTINUED WWW.FERTILITY MAGAZINE.ORG ON PAGE 38 • VOLUME 4 • FERTILITY MAGAZINE 37 AGE & FERTILITY C ONTINUED FROM PAGE 37 Menken J, Trussell J, Larsen U (1986) Age and infertility. Science 233, 1389-94. Munné S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, Cohen J, Sable D (2003) Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 7, 91-7. Pellestor F (2004) Âge maternel et anomalies chromosomiques dans les ovocytes humains. Med Sci (Paris) 20, 691-6. Piñón R (2002) ‘Biology of Human Reproduction.’ (University Science Books: Sausalito, CA, USA) Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman CW, te Velde ER (1999) Antral follicle counts by transvaginal ultrasonography are related to age in women with proven natural fertility. Fertil Steril 72, 845-51. Simpson JL, Elias S (1994) Prenatal diagnosis of genetic disorders. In ‘Maternal-fetal Medicine : Principles and Practice’. (Eds RK Robert K. Creasy and R Resnik) pp. 61-87. (W.B. Saunders: Philadelphia) Stachecki J, Cohen J (2004) An overview of oocyte cryopreservation. Reprod BioMed. Online 9, 152-163. Tarin JJ, Gomez-Piquer V, Rausell F, Navarro S, Hermenegildo C, 38 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG Cano A (2005) Delayed motherhood decreases life expectancy of mouse offspring. Biol Reprod 72, 1336-43. te Velde ER, Pearson PL (2002) The variability of female reproductive ageing. Hum Reprod Update 8, 141-54. U.S. Department of Health and Human Services - Centers for Disease Control and Prevention (2004) ‘2002 Assisted Reproductive Technology Success Rates: National Summary and Fertility Clinic Reports.’ Atlanta, GA, USA. U.S. National Center for Health Statistics (2003) Crude birth rates, fertility rates, and birth rates by age of mother, according to race and Hispanic origin: United States, selected years 1950-2002 .ftp://ftp.cdc.gov/pub/Health_ Statistics/NCHS/Publications/Health_US/hus04tables/ Table003.xls Date of access, July 2005. Wiemer KE, Anderson AR, Kyslinger ML, Weikert ML (2002) Embryonic development and pregnancies following sequential culture in human tubal fluid and a modified simplex optimized medium containing amino acids. Reprod Biomed Online 5, 323-7. Wilding M, Fiorentino A, De Simone ML, Infante V, De Matteo L, Marino M, Dale B (2002) Energy substrates, mitochondrial membrane potential and human preimplantation embryo division. Reprod Biomed Online 5, 39-42. You can contact Don Rieger, PhD at: [email protected] Introducing FertileAgeTM a unique and effective fertility supplement for both Women and Men. 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Female and male infertility may be caused by many factors such as environment, your habits, diet and age. Our fertility experts have explored the solutions and come up with the most effective fertility supplements in the market today. They utilize years of experience, scientific findings and published documentation to develop the concise balance of safe, proven, natural ingredients, and when taken daily may immediately improve your reproductive health. Daily doses of FertileAgeM supplements will provide your body with important scientifically proven ingredients that comply to our strict quality standards to enhance your reproductive system and your chances of conceiving. FertileAge M products are available worldwide through direct distribution and online channels and are designed to enhance the reproductive systems of women and men. www.FertileAge.com ©2005 FertileAge.com US/Canada: 800-720-6375 International: 519-826-5800 Fax: 519-826-6947 email: [email protected] FA-DISTAD-2005-05 PATIENT’S CORNER Exploration of Clinical and Laboratory Aspects of Infertility Series: Part 3 of 4 by Charlene Alouf, PhD, Laboratory Director, and Albert El-Roeiy, MD, MBA, Medical Director Crozer Chester Medical Center Fertility Center T he current topic for The Patient’s Corner is recurrent pregnancy loss or recurrent miscarriage. Nicole and her husband Jason have been married for 4 years and have been trying to have a child for the past CHARLENE A LOUF, PHD two. Nicole is a 35-year-old with a history of two first trimester miscarriages that occurred at 8 and 10 weeks of gestation. Nicole and Jason did not experience difficulty in conceiving either pregnancy. For the most part, Nicole ALBERT EL-ROEIY, MD, MBA has regular monthly menstrual cycles and there is no What are the causative factors? component of male factor infertility. A D&E was performed by Nicole’s gynecologist Factors attributed to habitual spontaneous abortions can following the second loss. During the procedure, Nicole’s be subdivided into genetic and non-genetic causes. The surgeon observed an abnormal structure, a wall dividing non-genetic factors include deviations in the uterine the uterine cavity called a septate uterus. Following the anatomy, infection of the reproductive tract, or surgery, the tissue from the pregnancy, or products of immunological or endocrine disturbances. Additionally, conception, was sent to a cytogenetics laboratory for a diseases associated with thrombotic complications may be chromosomal analysis (karyotype). Unfortunately, the responsible for recurrent loss. karyotype was inconclusive as the cells failed to grow in As a genetic basis for habitual abortions, numerical culture prohibiting a complete analysis. Therefore, the role chromosome abnormalities (aneuploidy) or structural of a possible contributory genetic abnormality could not aberrations (inversions, translocations, duplications be determined. The gynecologist referred Nicole and Jason and/or deletions) may be involved. Gonadal aneuploid to a reproductive endocrinologist for a second opinion on mosaicism (genetically unbalanced gametes in couples the uterine septum and to discuss the repeated pregnancy with a normal karyotype) must be considered. Single gene losses. At their consultation Nicole and Jason had many disorders may also contribute to recurrent losses. Most questions regarding their reproductive history and certainly, multiple etiologies may play a role in recurrent desired a plan of treatment to assist in achieving an loss as well as a host of unknown factors not currently ongoing pregnancy and live birth. established, as this list is non-exhaustive (reviewed in Simon et al., 1998, and Kearns et al., 2005). What defines recurrent pregnancy loss? The Inherited Pregnancy Loss Working Group defines What diagnostic tools are available to determine the recurrent pregnancy loss as 3 or more spontaneous losses cause of my repeated losses? occurring prior to fetal viability (<24 weeks gestation) Nicole and Jason were counseled regarding Nicole's (Laurino et al., 2005). However, it is not uncommon for history of subsequent pregnancy losses. As many factors medical professionals to commence evaluation of the impact reproductive wastage, Nicole and Jason couple after 2 such losses. Pregnancy loss is subunderwent a comprehensive baseline evaluation for characterized by gestational age, with loss occurring prior recurrent spontaneous abortion. The assessment included to 10 weeks defined as embryonic, while fetal loss defines that occurring at 10 weeks gestation and beyond. CONTINUED ON PAGE 42 WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 41 PATIENT’S CORNER C ONTINUED FROM PAGE 41 a thrombophilia workup that would identify factors that can cause clotting abnormalities. These include methyltetrahydrofolate deficiency, prothrombin and factor V Leiden gene mutations. These factors are involved with an increased frequency of venous thrombosis, or clotting events within the placenta, which result in pregnancy loss. Autoimmune studies, including anti-cardiolipin and antiphospholipid antibodies, are used to diagnose any immune reaction mounted against “self” tissue. Autoimmune disorders, or low titer antibodies which mimic autoimmune syndromes, may also contribute to pregnancy loss. Nicole’s thrombophilia workup and autoimmune study were both unremarkable. The impact of the endocrine system was also assessed during the baseline study. Abnormal hormonal levels could significantly impact the uterine environment and therefore, its function. Nicole’s androgen, progesterone, and prolactin levels were found to be within normal limits. Additionally, her thyroid function was normal, and she had no history of insulin-dependent diabetes. Although microbial infection as a causative agent in miscarriage is relatively low and controversial, mycoplasma and ureaplasma cultures were performed to exclude this as a factor. The results of the cultures were both negative. Confirming the gynecologist's suspicion, a septate uterus was identified by appropriate tests including a hysterosalpingogram and hysteroscopy. Additionally Nicole’s specialist recommended that a karyotype be performed on both her and Jason, in order to determine whether they were carriers of any chromosomal structural rearrangements, such as translocations, duplications and inversions. Healthy individuals can display balanced chromosomal structural rearrangements on karyotype analysis, meaning they contain the correct amount of genetic material. Balanced rearrangements can be passed on to healthy offspring or can become unbalanced during sperm or oocyte production. Embryos arising from the unbalanced gametes are at an elevated risk of growth arrest, implantation failure, and pregnancy wastage or the birth of a baby with a genetic syndrome. The karyotype analysis revealed that Nicole carries a balanced reciprocal translocation between chromosomes 9 and 10. She and Jason were referred to a geneticist for consultation to discuss the impact of these findings on past and future pregnancies. Does one factor outweigh the other with regards to the losses and what are our options for treatment? As habitual abortion represents a complex scenario 42 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG influenced by m u l t i p l e factors, it is difficult to discriminate between the contribution of either the u t e r i n e anomaly or the translocation per se, on an individual basis. However, due to Nicole’s translocation, she has at least a 50% chance of producing oocytes with a g e n e t i c imbalance. Because Nicole’s repeated losses might be the result of multiple etiologies, it was clear that the anatomical abnormality had to be addressed first. Nicole was in agreement and therefore a surgical procedure (hysteroscopy) with resection of the uterine wall was scheduled to correct the uterine anomaly. Nicole’s reproductive endocrinologist discussed additional technology available for her treatment – IVF with preimplantation genetic diagnosis (PGD). Recurrent miscarriage is just one diagnosis that appears to significantly benefit from IVF with PGD. The PGD process permits the identification of embryos that are affected with unbalanced chromosomal structural abnormalities or those which have an abnormal number of chromosomes (aneuploidy). The PGD procedure has an international misdiagnosis rate of approximately 5%. PGD offers the technology to transfer genetically balanced, normal embryos (for the probes tested) and thus increase the implantation rate and reduce the miscarriage rate. Nicole and Jason were awaiting a benefits election in her new employment position so they opted to delay entry into the IVF program for several months. Nicole proceeded with the hysteroscopy and within three months of the surgery she conceived again naturally. Unfortunately Nicole miscarried at 11 weeks of gestation. A karyotype was performed on the products of conception which revealed an unbalanced translocation (between chromosomes 9 and10) and also an XO karyotype (Turner’s Syndrome). After taking 5 months off from trying to conceive, the couple scheduled a return consult with the reproductive PATIENT’S CORNER endocrinologist to discuss starting an IVF/PGD cycle. Nicole and Jason were still in a good prognosis category as an IVF/PGD candidate despite their history of losses. They discussed aneuploidy screening in addition to that of the translocation due to the genetic evaluation of the previous pregnancy. Additionally, patients with a history of recurrent loss may have a higher frequency of aneuploid embryos during their IVF cycles. With this knowledge, Nicole and Jason elected to include aneuploidy screening in their PGD cycle. Nicole completed her IVF baseline testing and began stimulation. She demonstrated a very good response to her stimulation regimen; her follicular growth was well synchronized, and her estradiol level was appropriate for the number of mature follicles viewed on ultrasound. At the time of the retrieval, sixteen oocytes were identified. Fourteen oocytes were fertilized by conventional IVF, out of fifteen mature. The embryos developed well and were of good quality and by the third day of culture eleven embryos were available for biopsy. Two embryos were identified as genetically balanced and normal, based upon Nicole's translocation chromosome. These embryos were transferred and resulted in a single intrauterine pregnancy. Although Nicole and Jason were cautious throughout the entire pregnancy, Nicole gave birth to a full term healthy baby girl. In our next issue, we will address the treatment modalities available for couples diagnosed with severe male factor. Compromised sperm parameters not only affect fertilization, but also impact embryo development and implantation potential, and can contribute to pregnancy loss. References Laurino, MY, et al. Genetic evaluation and counseling of couples with recurrent miscarriage: recommendations of the national society of genetic couselors. J Genet. Couns. 14:165181, 2005. Simon, C, et al. Increased chromosomal abnormalities in human preimplantation embryos after in vitro fertilization in patients with recurrent miscarriage. Reprod. Fertil. Dev. 10:87-92, 1998. Kearns, WG, et al. Preimplantation genetic diagnosis and screening. Semin. Reprod. Med., in press. 2005. You can contact Charlene Alouf, PhD at: [email protected] and Albert El-Roeiy, MD, MBA at: [email protected] WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 43 PATIENT’S CORNER Helping You Understand “What Happens Next?” by Liz Sanders, Chief Embryologist Mississippi Fertility Institute, Jackson, Mississippi O ne of the key factors in resolving “uncertainty and fear: is communication. The IVF Team consists of the Reproductive Endocrinologist (R.E.), the Nurse Coordinator, and the Embryologist. It is imperative that you and the IVF Team communicate to get a better understanding of “what happens next” and what to expect. By the time you go to the R.E.’s office for your first visit, you have overcome many hurdles and crossed retrieval procedures, how to prepare for it, and what to expect. Also, the nurse will explain what happens next and how to prepare for the Embryo Transfer. They can also show you the room where everything will take place and the equipment used. This will help to resolve some of your anxiety about the procedures. Another member of the IVF Team is the Embryologist. The Embryologist can meet with you and explain everything that will take The basis of the correlation between age and infertility involves several factors. many bridges concerning infertility. But what lies ahead, and what can you expect? First, have all your questions written down, be brief and concise. You and the doctor will discuss what you have already been through and the tests that you have had done until now. At this point, the doctor may order more tests for you and your husband. One of the first tests he may order is a semen analysis for your husband, to be run in the clinic lab. You should receive a collection kit including written instructions concerning collection, transporting, and where to deliver the sample. Next, you will meet with another member of the IVF Team, the Nurse Coordinator. The nurse will sit down with you and explain your fertility plan in detail and go over instructions about your medications, when and how to take them, and when and where to have lab tests drawn and run. The nurse will explain to you about the egg place after your egg retrieval and leading up to the day of Embryo Transfer. The Embryologist will discuss with you the best procedure to use to achieve fertilization of your eggs. If the semen sample is considered below normal values, a procedure called Intracytoplasmic Sperm Injection (ICSI) will be performed. The Embryologist will actually select and pick up the best sperm and inject it into the cytoplasm of the egg. When lab tests are ordered you might hear acronyms used like P4, hCG, E2, and LH. Ask to have these test explained to you, what they stand for, and what YOUR results should be. Be sure the nurses have accurate information about how to get in touch with you concerning your lab results. Further, discuss if they can leave results and messages on your answering machine. This will save valuable time as the nurses make their calls each day. It is our goal as an IVF Team to LIZ SANDERS, CHIEF EMBRYOLOGIST answer all your questions and explain to you about In-Vitro Fertilization (IVF). We strive to treat our patients like people and not as numbers. We will help you understand “what happens next” in the process of achieving a pregnancy and experiencing the joys of parenthood. You can contact Liz Sanders, Chief Embryologist at: [email protected] WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 45 FEATURED PRODUCTS global ® global ® is a Simple and Flexible Embryo Culture Medium ONE solution for embryo culture from Day 1 through Blastocyst Services Performed: • PGD Diagnosis • Genetic Counseling for Patients • Embryo Biopsy & Embryology Services FDA Compliant US/Canada: 800-720-6375 International: 519-826-5800 Fax: 519-826-6947 [email protected] www.LifeGlobal.com Reprogenetics provides PGD analysis to IVF centers, including diagnosis of aneuploidy for advanced maternal age (such as Down’s syndrome), repeated IVF failure, recurrent spontaneous abortions, chromosome translocations and inversions, as well as for gene defects such as cystic fibrosis, fragile X, myotonic dystrophy, thalasaemia, Tay-Sachs, and others. We also provide embryologists that can perform embryo biopsy and cell processing. Contact Us At: www.reprogenetics.com Powder & Synthetic Latex Free Surgical Gloves Pristine gloves are completely free of talcs, starches, calcium carbonates or powdered substances or lubricants of any kind. Developed to protect the patient from granuloma, peritonitis, adhesions and other powder-related complications, Pristine gloves are also designed to protect against powder-related dermatitis and other powderassociated irritations of the hands and skin. US/Canada: 800-720-6375 International: 519-826-5800 Fax: 519-826-6947 [email protected] www.IVFonline.com 46 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG Tyho-Galileo Research Laboratories is an organization created by world-renowned specialists in reproductive science and molecular genetics. In founding the TyhoGalileo Laboratories, these individuals have joined together to stimulate progress in medical research, particularly in the areas of reproductive medicine, gametogenesis, human pre-implantation embryology and genetics. The research that Tyho-Galileo promotes will enhance the understanding of human reproduction and the origin of human disease and facilitate development of treatment modalities that can lead to the eradication of infertility and certain genetic diseases. Become a Galileo Center Contact: Jacques Cohen 973-322-6310 [email protected] www.galileocenters.com ALTERNATIVE MEDICINE Hypnosis for Childbirth by Shawn Gallagher, BA, CHt, Certified Hypnotherapist Midwifery Consulting Services T he staff in Labour and Delivery were stunned when they realized that the woman who presented was nine centimeters dilated and almost ready to push. It was her first baby and she had calmly laboured for a few hours at home before casually walking into the birth unit of the central Florida hospital her obstetrician had privileges in. Anna* was 29 years old and had seen me for a few hypnosis sessions in mid pregnancy to prepare for her baby’s birth. I first met her as a client of the Ontario Hypnosis Centre in association between hypnosis and better birth. Statistically significant outcomes of hypnosis include: faster labour, reduction in surgical delivery, less pain medication and shorter hospital stay. i-ix While individual response to pain in birth varies, women report a significant ability to manage the intensity of contractions without epidural or narcotic use. Additional research shows beneficial applications for nausea and vomiting in pregnancy (NVP), turning breech babies, reducing hypertension, …use the principles of hypnosis to promote a calmer, faster and more comfortable birth downtown Toronto where she had come for hypnosis to assist fertility. She had previously had an unsuccessful IVF cycle and was now looking at potentially reduced chances of pregnancy success with the use of frozen embryos. She had never been pregnant and now she wanted to improve her chances in any way she could. After four hypnosis sessions, she conceived a healthy pregnancy with her first frozen embryo transfer at a wellknown Toronto fertility clinic. Not long after conceiving, her husband received a work transfer to Florida and they moved there. She decided in mid pregnancy to return to Toronto for hypnosis as a way to encourage a natural birth. As a clinical hypnotherapist, I have trained over four hundred couples just like Anna and her partner to use the principles of hypnosis to promote a calmer, faster and more comfortable birth. Since hypnosis was accepted in 1955 by the American Medical Association, research in the medical uses of hypnosis clearly shows an insomnia, restless leg syndrome, smoking cessation and even threatened preterm delivery. High risk patients use hypnosis for preand post operative purposes with significant reductions in blood loss, infection rates and length of surgical time. The concern of potential negative effects of drugs on the fetus can result in some women avoiding physicianprescribed medication – hypnosis can be employed to encourage women to take their essential medication. Alternatively, women can also use hypnosis to calm themselves and even reduce their use of such prn prescriptions as anti-anxiety medication. Hypnosis encourages a very deep level of relaxation (not sleep) in even the most anxious, thus encouraging patient compliance and resulting in high satisfaction levels on the part of the woman, her partner and L&D staff. Women are always able to respond to the request of their attending nurse, midwife or physician while using hypnosis. In fact, women are in greater control of SHAWN G ALLAGHER, BA, CHT their responses and are significantly less likely to have a panic attack that can distract staff from their other important medical duties. “We have a simple method that decreases pain and anxiety and makes the invasive procedure safer and faster,” says Elvira Lang, M.D., director of cardiovascular and interventional radiology at Beth Israel Deaconess, Harvard Medical School in Boston, commenting on her study of the use of hypnosis as an adjunct in surgery. Nulliparous clients of the Hypnosis for Childbirth prenatal series report about half the length of labour as compared to the average population. In addition, these first time mothers have a caesarian section rate of 6.7 percent (vs an average of 20-25 percent) and an epidural rate of 18 percent (vs an average of 70-95 percent). Other reported effects include calmer babies postpartum and high rates of successful breastfeeding. Most women attending the four class Hypnosis for Childbirth series are highly anxious and list needles, hospitals, epidurals, caesarians and even doctors as among their worst fears. Stated goals at the first class WWW.FERTILITY MAGAZINE.ORG CONTINUED ON PAGE 48 • VOLUME 4 • FERTILITY MAGAZINE 47 ALTERNATIVE MEDICINE C ONTINUED FROM PAGE 47 include: being in control of one’s responses, being able to relax in a busy hospital setting, natural childbirth and feeling confident. Women and their partners are taught self-hypnosis, a variety of pain management tools and methods of self-induced deep relaxation. In today’s stress-filled culture, many are looking for alternatives for birth that are safe for them and safe for their babies. The safest method of pain relief is that achieved with hypnosis; the safest birth is a birth with a minimum of intervention. Anna’s birth proceeded smoothly. She was quickly ushered into the birth room and the attending physician was paged. As the birth equipment was laid out, she squatted with each push and rested between the pushes. She was able to relax deeply to her husband’s touch and voice as she was taught in the hypnosis sessions. As her doctor guided her, she was able to slow the delivery of the baby’s head and her alert baby was gently placed in her arms moments after the birth. The baby’s Apgar scores of 9/10 and 9/10 testified to the baby’s smooth transition into the world. The staff all commented on how smoothly the birth went and how unusual it was to see a birth with such a calm and relaxed patient. relaxation. Regardless of risk status, hypnosis has many other applications in obstetrics by reducing patient fears, encouraging comfort and improving the outcome of medical procedures. Medical professionals who wish to enhance patient comfort and satisfaction, contain health care costs and increase the incidence of natural childbirth can, with confidence, encourage their patients to use hypnosis. *a pseudonym References i Abramson, M., & Heron, W.T. An objective evaluation of hypnosis in obstetrics: Preliminary report. American Journal of Obstetrics and Gynecology, 59, 1069-1074, 1950. ii August, R.V. Obstetric hypnoanesthesia. American Journal of Obstetrics and Gynecology, 79, 1131-1137, 1960, and August, R.V. Hypnosis in obstetrics. New York: McGraw Hill, 1961. iii Brann LR, Guzvica SA. Comparison of hypnosis with conventional relaxation for antenatal and intrapartum use: A feasibility study in general practice. J R Coll Gen Pract 1987; 37:437-440. iv Hao TY, Li YH, Yao SF. Clinical study on shortening the birth process using psychological suggestion therapy. Zhonghua Hu Li Za Zhi. 1997 Oct; 32(10):568-70. (General Military Hospital of Jinan, P.R. China.) Summary Hypnosis is a focused form of concentration. Self-hypnosis is one form of hypnosis in which a certified practitioner teaches an individual to induce his or her own state of altered consciousness. Hypnotic analgesia is a safe and effective alternative and/or adjunct to pain medication. When used for childbirth pain, the primary aim of self-hypnosis is to help women maintain control by managing anxiety and discomfort through inducing a focused state of 48 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG v Harmon, T.M., Hynan, M., & Tyre, T.E. Improved obstetric outcomes using hypnotic analgesia and skill mastery combined with childbirth education. Journal of Consulting and Clinical Psychology, 58, 525-530, 1990. vi Hornyak, Lynne M. and Joseph P. Green. Healing From Within: The use of hypnosis in women's health care. Washington, DC: American Psychological Association, 2000. vii Jenkins, M.W., & Pritchard, M.H. Hypnosis: Practical applications and theoretical considerations in normal labour. British Journal of Obstetrics and Gynecology, 100(3), 221-226, 1993. viii Martin, Alice A., PhD; Paul G. Schauble, PhD; Surekha H. Rai, PhD; and R. Whit Curry, Jr, MD The Effects of Hypnosis on the Labor Processes and Birth Outcomes of Pregnant Adolescents. The Journal of Family Practice, May 2001, 50(5): 441-443. ix Mellegren, A. Practical experiences with a modified hypnosis-delivery. Psychotherapy and Psychosomatics, 14, 425-428, 1966. x Lang, Elvira et al. Adjunctive nonpharmacological analgesia for invasive medical procedures: a randomised trial. The Lancet, Vol 355, April 29, 2000, pages 1486-1490. xi Gallagher, Shawn. Hypnosis for Childbirth: A retrospective survey of birth outcome using prenatal selfhypnosis. Unpublished 2001 June; Toronto. You can contact Shawn Gallagher, CHt at: [email protected] HUMAN RESOURCES Full Time Embryologist Required The Fertility Center at Crozer-Chester Medical Center has a position available for a full time embryologist. The candidate must possess a B.S. with a minimum of 3 years experience in all facets of andrology and embryology. Preference will be given to those individuals with documented micromanipulation training. Alternating weekend coverage is required. CCMC is located ~20 minutes southwest of Philadelphia. US citizenship or permanent residency is required. Please contact Charlene A. Alouf, PhD, Laboratory Director, for additional information via email at [email protected] or call 610-447-2727. Visit our website at www.crozerfertility.com for additional information and our most recent CDC statistics. WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 49 RESOURCES The New England Fertility Institute is a patient oriented, full service treatment center, providing state-of-the-art, caring, fertility services. Dr. Gad Lavy and his trained team of fertility specialists are here to help you, by treating both female and male infertility with the most up-to-date techniques, in a modern facility. The New England Fertility Institute is consistently ranked among the top fertility clinics in the United States for the past 15 years. Come share in our success. New England Fertility Institute 1275 Summer Street, Stamford, CT, USA 06905 • 203-325-3200 9 Washington Avenue, Hamden, CT, USA 06518 • 203-248-2353 Visit our website at www.nefertility.com to see our success. Contact Dr. Lavy at [email protected]. We are conviently located close to New York City and major airports. 50 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG RESOURCES WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 51 CONFERENCES Delaware Valley Reproductive Biology Group Fall Dinner Lecture, September 7th, 2005 T he Delaware Valley Reproductive Biology Group held its fall dinner lecture on September 7th at the Red Sky Restaurant in Philadelphia. Members from Fertility Centers in Delaware, New Jersey and Pennsylvania were present to hear Dr. James Stachecki, Research Associate for Tyho-Galileo/Via-Cell Laboratories, present results from his ongoing oocyte cryopreservation project. The DVRBG members would like to express their sincere gratitude towards the generous sponsors of the evening’s event, Serono and Rosemont Pharmacy. I would like to thank everyone who attended, it was truly a wonderful evening! Charlene A. Alouf, PhD President, DVRBG Oocyte Cryopreservation: A Common Sense Approach James Stachecki, PhD O ocyte cryopreservation will be beneficial to women wanting to prolong reproduction in order to start a career, finish school, etc.; for egg donation purposes which could eliminate problems associated with donor-recipient synchronization and egg numbers; and for patients needing to undergo chemo- or radio-therapy. To date there is no reproducible method for freezing mature human eggs, although numerous children have been born (>100 children born worldwide, <5% pregnancy rate). Investigations over the past 20 years have failed to surpass, on a consistent basis, Chen’s 1986 report of the first child born from a frozen egg. This suggests that the current methodology is not adequate, and major changes need to be made in order to develop a successful, highly reproducible method for egg storage. Cryopreservation involves removing water from the cell by slow cooling in the presence of extracellular ice and disrupting the structure of the water that remains using a cryoprotectant(s) like PrOH, DMSO, EG, etc. For thawing, eggs are rewarmed at a rate slow enough to prevent thermal shock and rapid enough to prevent or minimize lethal ice crystal formation. Embryo storage has met with much greater success than egg storage and is used in IVF clinics throughout the world. Unfortunately, methods used for embryo storage do not work well for storing unfertilized eggs. In order to find a method that would allow successful egg storage, investigators have looked at the differences between eggs and embryos. Among these are microtubule and nuclear organization and membrane structure. There are numerous articles regarding spindle depolymerization and reformation upon cooling and rewarming, and as many that discuss differences in DNA structure (chromosomes vs. interphase nuclei), however these differences, although they could be responsible for possible post fertilization genetic errors, do not have a logical link with membrane intactness after thawing. In other words, survival rates should be near 100% even with spindle and/or chromosome disruption. On the other hand, membrane differences between eggs and embryos would explain the differences in survival rates after freezing. But do we really know why eggs survive cryopreservation poorly and what exactly are the causes of damage? Of the damage that could occur during cryopreservation, Intercellular Ice Formation (IIF) and osmotic effects are over-described in the literature as the major causes of damage. CONTINUED ON PAGE 54 However, there are other potential problems often overlooked, such as ion loading in WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 53 CONFERENCES response to cooling. As cooling progresses, energy driven processes will shut down. The Na/K antiporter is one of these processes driven by ATP and its functioning is responsible for maintaining a electrochemical balance inside the cell, necessary for normal cellular function. When this antiporter shuts down, sodium ions can leak inside the cell and load the cell with sodium, a condition that will exist upon thawing. The cell upon thawing “wakes up” to find that there is an overabundance of sodium ions that present a potentially toxic situation for the cell. Other reports only hint at the fact that IIF and solution effects may not present a problem when conventional cryopreservation procedures are used. We hypothesize that sodium loading along with freezing medium, cryoprotectant exposure, cooling, dehydration, thawing, and rehydration all present potential problems and need to be optimized in order to obtain 100% oocyte survival. We feel that IIF and solution effects, although potential problems to be aware of, are not the main cause for oocyte demise during cryopreservation. Early reports by our group demonstrated the effectiveness of removing sodium ions from the freezing medium and substituting these with choline ions. With mouse oocytes we achieved over 90% survival and 60% blastocyst formation rates in vitro, as well as pregnancy rates similar to control non-frozen oocytes. Preliminare experiments with human oocytes revealed that a low-sodium cholinebased cryopreservation medium (CJ3) was far superior to conventional sodium-based media. After protocol optimization along with modifications to the choline-based freezing medium, we obtained over 90% survival of fresh unfertilized human oocytes. These rates are higher than the best rates obtained with a high sucrose sodium-based media (Fabbri et al., 2001). Choline-based medium is being widely tested for its effectiveness in IVF clinics worldwide. CJ3 has significantly improved the already good results obtained with CJ2 when freezing mouse and human oocytes. At least 5 babies have been born worldwide using CJ3 and over 15 babies using a low-sodium choline-based medium. In addition to slow-cooling protocols, vitrification methods have recently become popular, but they remain to be perfected for use with oocytes. We present here preliminary results when vitrifying blastocysts with a newly developed method called: S3-vitrification. As more and more clinics convert to blastocyst transfer to reduce multiple pregnancies, a safe, efficient, and reproducible method for storing blastocysts will be needed. S3-vitrification technology fills this need. This is a new method that avoids the problems associated with other vitrification methods currently used and is an effective, reproducible, and user-friendly method for storing human blastocysts. S3-Vitrification does not use DMSO, a potentially toxic cryoprotectant used in other vitrification procedures. Blastocysts are frozen in conventional heat-sealed 0.25cc straws eliminating direct contact with LN2 and thereby helping to avoid potential bacterial and viral contamination. There are no micro-sized straws, loops, or grids to mess with in our system. No blastocoel collapsing or reducing is required for maximal survival, and all blastocyst stages, from expanding to fully hatched, can be frozen with this method. S3-vitrification employs longer exposure and loading times (up to 3 time longer) which leaves room for possible technician-related errors when trying to expose, load, and freeze blastocysts in 45 seconds or less, the typical time constraint of other methods. Preliminary results have provided over 90% survival and re-expansion of any stage blastocyst, including >90% survival of ICM & trophectoderm cells. We have preliminary clinical reports of ongoing survival rates of over 90% and ongoing pregnancy rates of over 60%. For more information regarding S3-vitrification please contact Kelly at: [email protected] or (973) 322-6315 C ONTINUED FROM PAGE 53 54 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG CONFERENCES WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 55 CONFERENCES XVIII FIGO Contact US Tel: +60 4252 9100 Fax: +60 4257 1133 email: [email protected] www.figo2006kl.com 56 FERTILITY MAGAZINE • VOLUME 4 • WWW.FERTILITYMAGAZINE.ORG World Congress of Gynecology and Obstetrics Kuala Lumpur, Malaysia November 5-10, 2006 Kuala Lumpur Convention Centre CONFERENCES 5th MSRM ANNUAL MEETING Contact Information: MSRM Coordinator Dr. Ashraf Samir [email protected] PO Box 125, Ibrahimieh Alexandria, Egypt 21321 Tel. & Fax: +20 3 359 50 43/44 Congress Secretariat ERA Ltd. 8, Alex. Soutsou str. 106 71 Athens, Greece Congress Website www.msrmcrete2006.com WWW.FERTILITY MAGAZINE.ORG • VOLUME 4 • FERTILITY MAGAZINE 57 I’ve planned for this my entire life. 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