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Volume 2, Issue 6
Winter 2001 M E D I C A L
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The Use of Fluorescent Dyes In ART Amy Jones, BS Reproductive Biology Associates, Altanta, GA Director of the Preimplantation Genetic Diagnosis Program at RBA INTRODUCTION
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luorescent dyes allow researchers to detect specific components of living and fixed cells with acute sensitivity. A fluorophore or fluorescent dye is designed to react to a specific stimulus within a localized region of a biological specimen. Energy supplied from an external source such as a mercury bulb or laser is absorbed by the fluorophore generating what is called an excited state, or fluorescence. Fluorescent detection requires four fundamental components: (1) an excitation source, (2) a fluorophore, (3) wavelength filters, and (4) an imaging system. Fluorescent dyes have historically been an important tool in understanding the dynamics of oocyte development, sperm integrity, and embryo quality. As early as 1979, fluorescent staining allowed investigators to examine sperm surface components after fertilization; thus gaining a better understanding of the cell fusion system at fertilization in the mouse and the sea urchin (Gabel et al. 1979). Recently however, the use of fluorescent dyes has become a fundamental component of many human IVF labs since the introduction of preimplantation genetic diagnosis (PGD) using fluorescent in situ hybridization (FISH). Fluorescent staining has also been used to examine mitochondrial distribution, DNA integrity, polarized distribution of Continued on Page 5
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The Embr yologists’ Newsletter
Winter 2000
Vitrification of Embryos Using a Nylon Loop Michelle Lane, PhD Research and Development Colorado Center for Reproductive Medicine 799 East Hampden Ave., Suite 300 Englewood, CO 80110 INTRODUCTION
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he ability to cryopreserve embryos is paramount to a successful IVF program especially with the introduction of blastocyst culture resulting in a reduction in number of embryos transferred. In recent years there have been significant advances in the area of cryobiology for storage of animal oocytes and embryos where there has been a steady move towards vitrification as the procedure of choice (Kasai, 1997). Most significantly the development of ultra-rapid vitrification procedures such as open-pulled straw and loop vitrification have resulted in significant improvements in the ability to cryopreserve animal oocytes and embryo. In human IVF, embryos have been traditionally cryopreserved using slow-freezing protocols employing low concentrations of cryoprotectants coupled with slow controlled rates of cooling. During slow-cooling protocols the cells shrink and dehydrate as the concentration of solutes increases as water freezes in the medium. This prevents the formation of intracellular ice crystals (Leibo et al., 1978). However, currently even in the best programs the viability of frozen/thawed human embryos is significantly less than the fresh embryos. An alternative to slow freezing is vitrification.
Vitrification uses very high concentrations of cryoprotectants which when rapidly cooled results in the sample being solidified by an extreme elevation in viscosity and is often referred to as a glasslike state. Vitrification avoids crystallization therefore eliminating the possibility of intracellular ice crystal formation. ULTRA RAPID VITRIFICATION
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ecently there has been the development of ultra-rapid vitrification procedures. The first report of ultra rapid vitrification was from Dr. Leibo and colleagues who described a vitrification system using an electron microscope grid (Martino et al. 1996). This open system increases the rate of heat transfer, thereby reducing the toxicity of the cryoprotectants. Using this procedure bovine oocytes could be successfully cryopreserved. Subsequently, Dr. Gabor Vajta also reported successful vitrification of bovine oocytes using an open pulled straw (Vatja et al., 1997). This technique uses straws with thinner walls to increase the rate of heat transfer. NYLON LOOP VITRIFICATION
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imilar to the principles of these techniques, vitrification using a nylon loop was reported (Lane et al. 1999a).
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Oocytes and embryos are suspended in vitrification solution in a small nylon loop of between 0.20.5mm in diameter. The loop containing the embryos is then plunged into a cryovial filled with liquid nitrogen and the cells vitrified. This system lacks a thermoinsulating layer thereby maximizing the rate of heat transfer. The benefits of this procedure are that the manipulations involved are very simple and either a single or multiple embryos can be placed on a single loop. Vitrification using the nylon loop has been shown to be versatile and has been used to successfully vitrify embryos at different stages from rodents (mice, hamster and rat), domestic animals (cow, horse) and primates (Table 1). There have also been several successful pregnancies established in rodents and domestic animals. While there has been a preliminary report on the in vitro development of leftover poor quality human blastocysts, there has been little research on the use of the nylon loop for the vitrification of human oocytes or embryos. The data reported for animal embryos are very encouraging. However, the efficacy of the loop vitrification on human embryo cryopreservation awaits testing using a prospective randomized trial. Continued on Page 6
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certain proteins and other oocyte and embryonic traits that might help scientists better understand mechanisms involved in production of normal offspring. FISH
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n 1989 Penketh et al sexed human blastomeres by FISH with either X or Y probes. Griffo et al. (1990) sexed mouse embryos by simultaneous FISH with X and Y probes. PGD was still out of reach at this stage, because the time requirements for FISH were not compatible with the IVF cycle. In 1993 Munne et al were able to sex human embryos by FISH within a day using directly-labeled probes for X and Y simultaneously (Munné et al. 1993a), and soon after (1993b) they were able to simultaneously analyze chromosomes X, Y, 18, 13, and 21 in blastomeres by FISH. This new FISH timetable realistically opened the door to PGD for aneuploidy during a fresh IVF cycle. Twenty-one percent of spontaneous abortions are caused by numerical chromosome abnormalities, and the main risk factor is maternal age with trisomies increasing from 2% in women 25-years-old to 19% in women over 40 (Warburton et al. 1986). According to ASRM-SART data (1998) 52% of IVF stimulation cycles in the USA are carried out in women 35 years or older, demonstrating how IVF patient populations would greatly benefit from the screening of chromosome aneuploidies by FISH. It was hypothesized that negative selection of aneuploid embryos would improve implantation rates, because of the correlation between
advanced maternal age and chromosomally abnormal embryos (Munné et al. 1993c). One of the main advantages of FISH is that it doesn’t require cell culturing or preparation of metaphase spreads, as is required for karyotyping. Currently FISH has been applied to PGD of chromosomal abnormalities in X, Y, 13, 14, 15, 16, 18, 21 and 22 (Munné et al., 1998a). This accounts for 70% of aneuploidies detected in spontaneous abortions. FISH analysis has been used for preconception diagnosis by using the first and second polar bodies as an indicator of the genetic status of the oocyte (Verlinsky et al. 1990, 1998). A disadvantage of polar body analysis alone is that it does not take into account paternal aneuploidies. Preimplantation genetic diagnosis of translocations involves a slightly more complex FISH technique. Polar bodies can be used for the analysis as more than 90% of 1st polar bodies fixed within 6 hours after retrieval are in metaphase (Munné et al. 1998c). The fluorescent probes used for this technique include whole chromosome painting probes, telomeric probes and centromere probes to differentiate chromatids. If the FISH results are still ambiguous, then embryo biopsy may be required to confirm the diagnosis. Unlike polar bodies, blastomeres from an 8-cell embryo are usually in interphase, making whole chromosome painting impossible. Thus, the biopsied cells must be brought into metaphase. Willadsen et al. (1999) diagnosed translocations by fusing a single blastomere from a Day 3 embryo with a bovine oocyte. This process forces the human nucleus into metaphase
making whole chromosome fluorescent painting possible. The aforementioned techniques either limit diagnosis of translocations to female carriers or they require the complex procedure of fusing cells. Recently a new technique for translocation detection has been developed which involves the use of telomeric probes and can be carried out on interphase blastomeres (Munne et al. 2000). Thus, translocations of either parental origin can identified. ASSESSMENT OF OOCYTE AND EMBRYO QUALITY
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etrieval of immature oocytes is a common occurrence in a stimulated IVF cycle, and these germinal vesicle and metaphase one stage oocytes are often cultured overnight, which frequently leads to maturation and insemination by conventional methods or ICSI. Park et al. (1997) demonstrated by FISH that human oocytes that were matured in vitro after cryopreservation at the germinal vesicle stage, displayed an increased frequency of chromosomal abnormalities, which could adversely affect the developmental potential of embryos derived from cryopreserved oocytes. Mammalian oocytes of all the species studied thus far display a series of intracellular calcium (Ca2+i) elevations during fertilization, which are responsible for triggering the activation of metaphase II oocytes (Miyazaki et al. 1993). Monitoring of intracellular Ca2+ can be done with fluorescent dyes. Kono et al. (1996) demonstrated that there is an endogenous increase in Ca2+ that produces Ca2+ transients specifiContinued on Page 11
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Table 1. Summary of success with nylon loop vitrification Species Mouse1 Hamster2 Rat2 Cow2 Non-human primate3
Oocytes
Blastocysts
Yes Yes nd Yes
Cleavage Stage Embryo Yes Yes Yes Yes
nd
nd
Yes
Yes nd nd Yes
nd- not determined. 1Lane et al., 1999b, 2Lane et al., 1999a, 3Yeoman et al., 2000.
USE OF THE NYLON LOOP FOR THE VITRIFICATION OF MOUSE EMBRYOS METHODS mbryos are placed in a Hepes-buffered medium containing 8% ethylene glycol and DMSO for between 1-3 minutes (1 minute for zygotes, 3 minutes for blastocysts). Embryos are then transferred to a second cryoprotectant solution containing 16% ethylene glycol and DMSO with 0.65M sucrose and ficoll for
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around 30 seconds. Embryos are then transferred to a 0.5mm diameter loop and plunged directly into a cryovial containing liquid nitrogen. For thawing, embryos are thawed into a Hepes-buffered medium containing 0.5 M sucrose for 1-2 minutes (1 minute for zygotes, 2 minutes for blastocysts), then 2-3 minutes in a medium containing 0.25M sucrose (2 minute for zygotes, 3 minutes for blastocysts) and then 5 minutes in a Hepes-buffered medium. Embryos can then be placed into culture.
RESULTS AND DISCUSSION
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here is no difference in the ability of mouse 1-cell or 2cell embryos to develop to the blastocyst stage in culture following vitrification using the nylon loop (Table 2). Blastocysts resulting from vitrified embryos had equivalent cell numbers as control blastocysts that were not frozen. There appears to be no loss in developmental competence of mouse embryos vitrified on a nylon loop.
Table 2. Effect of vitrification on mouse embryo development to the blastocyst stage Stage at Collection
Treatment
Zygote Zygote 2-cell 2-cell
Control Vitrified Control Vitrified
Blastocyst Development (%) 92 94 98 95
Blastocyst Cell Number 75.4±1.2 73.8±2.1 85.7±2.2 88.7±2.7
Embryos were cultured in G1.2/G2.2 media to the blastocyst stage (96h to zygote, 72h for 2-cell embryos). N=100 per treatment
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Understanding Embryology Costs Stan Colquitt, President Medical Market Analysts (This is the first in a series of articles designed to help embryologists understand laboratory costs.)
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eproductive medicine practices, while primarily concerned with successful treatment outcomes, must understand their "costs." Profit is a function of cost and includes labor, materials, and fixed and variable overhead costs. Knowing and understanding these costs are the first steps to establishing appropriate prices. This information is critical to negotiating managed care contracts, which often have very low profit margins. Embryology procedures are a major component of overall ART costs. In practices that we have analyzed, embryology is also the most profitable department. Given this assertion, laboratory cost analyses of high-volume procedures (highest revenue contributors) will yield information that is essential to controlling costs and establishing prices. We divide the practice into profit centers, such as Andrology, Embryology, Endocrine, Reproductive Medicine, etc. The revenue
for each center is ascertained by examining CPT charges for the preceding calendar year. Knowing the revenue of each profit center allows overhead to be assigned to departments in relation to their contribution to revenue. Overhead is a major component of cost and must be distributed across all procedures. We establish this distribution based upon each procedure’s relative contribution to revenue. For example, if Embryology contributes 60% of revenue, it will carry 60% of overhead costs. If ICSI comprises 30% of embryology revenue, it will carry 30% of the Embryology Department's overhead costs. Overhead by procedure is determined by dividing total overhead for each CPT code by the number of procedures conducted. This relationship readily demonstrates the manner in which increased volume lowers cost per procedure when the majority of overhead is "fixed." This is the rationale sometimes used for accepting contracted procedures at or slightly above cost. Obviously,
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costs must be known to ensure reasonable profitability. To begin your analyses, ask the office manager for a list of embryology CPT codes with associated revenue for the last calendar year. Ideally, these data can be downloaded into a database, such as Excel, which can be easily sorted and manipulated. Determine total practice and Embryology Department revenue (based upon aggregate CPT codes). Once these data are known, total overhead data can be obtained from the office manager. I will discuss overhead allocation in the next issue. If you have questions, please do not hesitate to contact me at (704) 598-9309 or through www.embryologists.com.
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SUMMARY
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ylon loop vitrification of mouse embryos does not affect developmental competence. However, the permeability of oocytes and embryos differs both between species and between stages. Therefore, subtle but important differences may exist in protocols required to successfully vitrify oocytes and embryos from different species. However, the apparent versatility of this loop vitrification procedure for animal embryos gives promise that this technique could be developed into a reliable method for human oocyte and embryo cryopreservation.
REFERENCES 1) Kasai, M. (1997) Cryopreservation of mammalian embryos. Mol Biotechnol. 7,173-9. 2) Lane, M., Forest, K.T., Lyons, E.A., Bavister,B.D. (1999a) Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol.7, 1234-6. 3) Lane, M., Schoolcraft, W.B., Gardner, D.K. (1999b) Vitrification of Mouse and Human Blastocysts Using a Novel Cryoloop Container-less Technique. Fertil. Steril., 72, 1073-8. 4) Leibo, S.P., McGrath, J.J., Cravalho, R.G. (1978) Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate. Cryobiology, 15, 257-271.
5) Martino, A., Songasen, N., Leibo, S.P. (1996) Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod., 54, 1059-1069. 6) Vajta, G., Booth, P.J., Holm, P., et al. (1997) Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. CryoLetters, 18, 191-195.\ 7) Yeoman, R.R., Gerami-Naini, B., Nusser, K.D., et al. (2000) Modified Cryo-Loop vitrification yields superior post-thaw survival and development of rhesus monkey blastocysts. Fertil. Steril., 74 (suppl 1), O-121.
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Dr. Piquette Discusses Possible Links Between ART Therapy and Birth Defects Gary N. Piquette, PhD, HCLD Northridge Center for Reproductive Medicine 18546 Roscoe Blvd. Suite 240 Northridge, CA 91324 (818) 701-8181 "Dr. Piquette discusses several factors that must be considered when examining the possible links, if any, between ART therapy and fetal birth defects. We encourage your comments and contributions to this forum in upcoming issues of the newsletter."
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here has been much discussion concerning the use of superovulatory hormones in ART procedures and their potential for inducing fetal birth defects. One
source of information on this subject for patients undergoing ART in the United States is the yearly report of the Society of Assisted Reproductive Technologies (SART) in conjunction with the Center For Disease Control (CDC) published in the journal Fertility and Sterility. If one looks back several years, the birth defect rates for patients treated with ovulation inducing agents are not statistically different from the general population. The birth defect rates for IVF alone
(6,379 deliveries) or IVF plus ICSI (3,632 deliveries) were both 1.8% of neonates born in the United States in 1996 (Fertil Steril 1999;71:798-807). The specific birth defects or causes were not reported. In the United States in 1997, the birth defect rates for IVF alone (7,353 deliveries) or IVF plus ICSI (4,949 deliveries) were 1.6% and 1.7% respectively, of neonates born in each group. (Fertil Steril 2000;74:641-654) Again, the speContinued on Page 10
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cific birth defects or causes were not reported. In 1996, the incidence of triplets or greater resulting from fresh, nondonor ART cycles was less than 6%; twin births accounted for about 26%, whereas single births were about 52%. The remaining outcomes were still births, ectopic pregnancies or abortions (induced and spontaneous). Birth defects could be a result of the stresses and difficulties of a multiple pregnancy be it natural, ART, or IUI. One needs to determine specifically if ART is a causative factor. IUI patients are often treated with superovulatory hormones and once pregnant with multiples may not accept fetal reduction as a means to decrease higher order birth complications. For these and other reasons, the incidence of multiple pregnancies and births in IUI patients are probably higher than those in ART patients. Yet, this cannot be established conclusively because these data are not reported to SART or the CDC. We control the number of embryos transferred to the uterus with ART patients and thus limit potential problems, such as birth defects due to multiple pregnancies. As previously stated, it is more difficult to control the multiple rates in IUI patients. Another factor that can contribute to birth defects is female patient age. As women age, there is a higher incidence of birth defects and miscarriages, likely due to oocyte chromosomal problems. Therefore, a study of potential ART related birth defect rates should consider that ART patients are often in these higher age groups thus raising the median treatment age. For this reason, it is necessary to compare birth defect rates to similar age groups in the untreated population. One would also need to discern possible causes due to the actual ART procedure versus those resulting from delivery procedures, prenatal and postnatal care as well as genetic and environmental factors. Several papers regarding birth defects due to intracytoplasmic sperm injection (ICSI) alone or ICSI plus testicular epididymal sperm extraction (TESE) have been reported by Andre Van Steirteghem's group in Belgium and they have warned of possible problems with offspring. Although offspring resulting from ICSI using poor quality sperm or low numbers of sperm (such as those acquired directly from the epididymis or testicle) may not have a specifically recognized birth defect, these offspring could be carrying a gene defect (such as a Y deletion) which may render the male offspring infertile.
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cally during mitosis. Furthermore, using Calcium Green-1 dextran indicator dye Wilding et al. (1996) discovered that calcium increases in the perinuclear region of sea urchin embryos initiate the entry into mitosis. Studies using fluorescence (Calcium Green-1) have also indicated that there is a specific intracellular calcium release along the cleavage furrow during cytokinesis (Muto et al. 1996). The artificial conditions used for IVF culture can be stressful to oocytes and embryos, inducing anomalies such as zona hardening and fragmentation. Using immunofluorescence Antczak et al. (1999) were able to evaluate the relationship between the temporal and spatial characteristics of fragmentation and the distribution of certain regulatory proteins. Previous studies using immunofluorescent staining established the polarized distribution of leptin and STAT3 as early as the 2-cell stage (Antczak et al. 1997), and the current study confirmed that additional regulatory proteins were peripherally located. Thus during development different regions of the embryo are either rich in regulatory proteins or depleted, and this condition persists throughout the early embryonic stages; the depleted regions finally contributed to the inner cell mass (ICM) of the blastocyst while regulatory protein rich and deficient cells formed the trophectoderm. However, when a 2-cell, 4-cell or 8-cell embryo forms fragments, the loss of regulatory proteins is likely due to their peripheral location, thus demonstrating the possible deleterious effects of fragmentation. Various causes of fragmentation have been suggested. The in
vitro culture environment has a higher oxygen concentration than the in vivo environment (Yang et al. 1998). In such conditions embryos produce reactive oxygen species (ROS), which are believed to cause damage to cell membranes. In order to determine the morphological characteristics of cell damage induced by H2O2, investigators measured the relative intensity of H2O2 concentrations within embryos using 2',7'dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging. H2O2 concentrations were significantly higher in fragmented embryos versus nonfragmented embryos and unfertilized oocytes (Yang et al. 1998). Apoptosis is a significant concern when culturing human oocytes and embryos in vitro and is often associated with fragmentation. A cell may generate fragments and still continue to divide (Antczak et al. 1999), which is clearly not indicative of apoptosis. Along the same lines, DNA strand breaks were examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) and phosphatidylserine by annexin V staining, with imaging by epifluorescence and scanning laser confocal fluorescence microscopy. The investigators determined that despite previous studies, indicating that the detection of 3’OH DNA strand breaks by TUNEL in mouse oocytes is evidence for apoptosis, their studies found no evidence to support this hypothesis. Rather their findings suggest that annexin V staining of some cytoplasts in fragmented oocytes is merely an indication of compromised plasma membrane, and the pattern of
chromosomal TUNEL fluorescence and its incidence in only some MII human chromosomes suggests that this probe could be picking up DNA stand breaks that occurred before the resumption of meiosis and thus are not signs of apoptosis. (Van Blerkom, 1998) Scoring of 2pn zygotes has recently become a useful method of identifying good quality embryos early in development (Scott and Smith, 1998). While some investigators focus on nucleoli alignment (Tesarik et al. 1999) or the differential “halo effect” observed in some fertilized eggs (Scott and Smith, 1998), Manor et al. (1999) used FISH to detect chromosome anomalies in embryos with unequal sized pronuclei. They determined that chromosome anomalies were present in 88.5% and 50% of embryos derived from unequal sized pronuclei in ICSI and conventionally inseminated embryos respectively. CONCLUSIONS
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he use of fluorescent dyes has facilitated a better understanding of gamete and early embryo potential. They have even played a role in the cloning procedure. One of the initial steps in the cloning process requires the removal of maternal chromosomes to produce an enucleated oocyte. To visualize the metaphase spindle and verify the complete removal of its components the investigators used fluorescence (Dominko et al. 2000). There are many applications for fluorescent dyes in the field of reproductive medicine that allow scientists to observe aspects of gamete and embryo development Continued on Page 15
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that could not otherwise be seen. The reproductive scientist of today needs to have a basic understanding of staining principles for designing experiments or even reading articles that use the techniques.
5. Grifo JA, Boyle A, Fischer E, Lavy G, DeCherney AH, Ward DC, Sanyal MK (1990) Preembryo biopsy and analysis of blastomeres by in situ hybridization. Am.J.Obstet Gynecol 163:2013-2019
REFERENCES
6. Handyside AH, Kontogianni EH, Hardy K, Winston RML (1990) Pregnancies from biopsed human pre-implantation embryos sexed by Y-specific DNA amplification. Nature 344,768- 770
1. Antczak, M. and Van Blerkom, J. (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT 3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol. Hum. Reprod., 3, 1067–1086 2. Antczak, M. and Van Blerkom, J. (1999) Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum. Reprod., 14, 429–447 3. Dominko T, Chan A, Simerly C, Luetjens CM, Hewitson L, Martinovich C, Schatten G. (2000) Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidance of parthenogenesis, and successful embryogenesis. Bio Reprod Jan;62(1):150-4 4. Gabel CA, Eddy EM, Shapiro BM. (1979) After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos. Cell Sep;18(1):207-15
7. Kono T, Jones KT, BosMikich A, Whittingham DG, Carroll J. (1996) A cell cycleassociated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division. J Cell Biol Mar;132(5):915-23 8. Manor D, Drugan A, Stein D, Pillar M, Itskovitz-Eldor J. (1999) Unequal pronuclear size--a powerful predictor of embryonic chromosome anomalies. J Assist Reprod Genet Aug;16(7):385-9 9. Miyazaki, S., Shirakawa, H., Nakada, K. and Honda, Y. (1993) Essential role of the inositol 1,4,5 trisphosphate receptor/ Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian oocytes. Dev. Biol., 158, 62–78 10. Munné S., Weier H.U.G., Stein J., Grifo J., Cohen J. (1993a) A fast and efficient method for simultaneous X and Y in situ hybridization of human blastomeres. J. Assisted Reprod. Genet. 10: 82-90
11. Munné S, Grifo J, Cohen J (1993b) Preimplantation genetic diagnosis with fluorescence in situ hybridization. Assisted Reprod.Reviews 3,2:100-106 12. Munné S, Grifo J, Cohen J (1993c) Preimplantation genetic diagnosis with fluorescence in situ hybridization. Assisted Reprod. Reviews 3,2:100-106 13. Munné S, Márquez C, Reing A, Garrisi J, Alikani M (1998a) Chromosome abnormalities in embryos obtained following conventional IVF and ICSI. Fertil Steril 69:904-908 14. Munné S, Scott R, Sable D, Cohen J (1998b) First pregnancies after preconception diagnosis of translocations of maternal origin. Fertil Steril 69:675-681 15. Munné S, Magli C, Bahçe M, Fung J, Legator M, Morrison L, Cohen J, Gianaroli L (1998c) Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenatal Diagn 18:1459-1466 16. Munne S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J. (2000) Outcome of pre-implantation genetic diagnosis of translocations. Fertil Steril Jun;73(6): 1209-18 17. Muto A, Kume S, Inoue T, Okano H, Mikoshiba K. (1996) Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin. J Cell Biol. Oct;135(1):181-90. Continued on Page 18
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Other factors to consider are the superovulatory hormones being used, especially with several preparations on the market. Since natural cycles are rarely, if ever, used in ART procedures, it must be assumed that most ART patients are receiving combinations of FSH, LH, and GnRH antagonists or agonists. Furthermore, many patients are treated with many forms of progesterone supplements. In order to study the possible relationship(s) between superovulatory hormones and birth defects, one must be able to separate the effects due to drug combinations and varying doses of each of these hormones. This is a very difficult task and requires strict controls and large numbers of patients. I wouldn't discourage anyone from devising a study to answer the question: Does ART specifically cause an increase in distinct birth defects? One must be careful not to draw conclusions of cause and effect without sufficient data. Since treatments are constantly changing in ART and birth defect rates are low, studies will require very large number of patients in several different treatment groups (hormone treatment, age, single birth, twin birth, triplets or more, egg donor, ICSI, etc.) to arrive at statistically meaningful analyses. SART has attempted to address these issues by collecting more data through changes in reporting criteria. But as we all know, the yearly increases in specific data reporting on various topics has created a huge labor problem in many clinics. One must be careful to maintain balance between collecting data and providing patient care. Collecting data is costly to any laboratory and it should yield information that can improve clinical
practice. It would be virtually impossible to include all the current and future (unrecognized) birth defects in any reporting scheme. Given the low percentage of total birth defects, identification of a specific defect would be impractical (or impossible) considering the relative number of patients receiving ART therapy. We counsel our patients, and include on all consent forms, statements such as "Although we don't know what specific birth defects they are at risk for with ART, the overall birth defect rate for the last several years, as reported by SART and CDC, is not statistically different than that of the general population.” Patients undergoing ICSI are informed specifically that their offspring are especially at risk for specific gene defects that might render their children infertile as well. Although we recognize that males produced by ICSI are especially at risk for infertility, little if anything is known of the incidence of specific problems for female offspring produced by ICSI.
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n conclusion, in treating the infertile couple we have acquired many tools including the use of superovulatory hormones, alternative methods of acquiring sperm and micromanipulation techniques such as ICSI. There does not seem to be a difference in birth defect rates compared with those found in natural cycles. However, there are many factors, which need to be analyzed in order to discern that specific defects might be higher in ART offspring. It is our responsibility as health care providers to reveal as much information to our patients as we can regarding their risks, not just to themselves but to their offspring.
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This information should be part of the informed consent document that the patients sign. If we are to provide more information regarding risks such as birth defects, we must continuously analyze outcomes of our treatments. But by the same token, we cannot conduct studies without the proper controls or comparisons. It would be far more damaging to our field to provide erroneous information to our patients based on improper analysis and interpretation of data than to simply say we don’t know what the risks are and proceed with caution.
❖ Position Announcement Washington Center for Reproductive Medicine Bellevue, Washington We are seeking an experienced embryologist to supervise a state of the art new IVF laboratory in Bellevue, Washington. Bellevue is a beautiful city located outside of Seattle. Candidates should be highly accomplished in all aspects of IVF including ICSI and have the ability to develop the laboratory to its full potential. Familiarity with accreditation procedures, including CAP, required. Competitive salary plus generous incentives. Please fax resume to (425) 688-7684.
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18. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. (1997) Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril Nov;68(5):920-6
24. Verlinsky,Y., Cieslak,J., Ivakhnenko,V. et al (1998) Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis. J. Assist Reprod.Genet., 15, 285-289. 25. Warburton D, Kline J, Stein Z, Strobino B (1986) Cytogenetic abnormalities in spontaneous abortions of recognized conceptions. In: Porter IH, Willey A, eds. Perinatal Genetics: Diagnosis and Treatment. New York: Academic Press, 1986:133-148
19. Penketh RJA, Delhanty JDA, Van den Berghe JA, Finklestone EM, Handyside AH, Malcolm S, Winston EML (1989) Rapid sexing of human embryos by nonradioactive in situ hybridization: potential for preimplantation diagnosis of X-linked disorders. Prenat. Diagn. 9, 489-500
26. Wilding M, Wright EM, Patel R, Ellis-Davies G, Whitaker M. (1996) Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos. J Cell Biol. Oct;135(1):191-9
20. Scott, L.A. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following the oocyte retrieval. Hum. Reprod. 13. 1003-1013.
27. Willadsen S, Levron J, Munne S, Schimmel T, Marquez C, Scott R, Cohen J. (1999) Rapid visualization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum Reprod Feb;14(2):470-5
21. Tesarik, J., Greco, E., (1999) The probablility of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum Reprod., 14, 1318-1323 22. Van Blerkom, J, David, PW. (1998) DNA strand breaks phosphatidylserine resfistribution in newly ovulated and cultured mouse and human oocytes: occurrence and relationship to apoptosis. Hum Reprod May;13(5):1317-1324
28. Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. (1998) Detection of reactive oxygen species (ROS) and apoptosis in human frag-mented embryos. Hum Reprod Apr;13(4):9981002
23. Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM 1990) Analysis of the first polar body: preconception genetic diagnosis. Human Reprod. 5:826-829
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The Embr yologists’ Newsletter
Winter 2000
Ensure That Your Web Site Design is Optimized for Search Engines Cliff Bernstein, Ph.D. President, Internet Health Resources http://www.ihr.com Note: this article is the first in a series of articles written to help infertility organizations promote their infertility Web sites on the Internet. rankings. Web site popularity is now a very important factor for search engine rankings.
I
f you build your infertility Web site with an understanding of how search engines work, it will be more likely that your site will rank high in the search engines. Internet Health Resources1 provides the following tips to help you optimize your Web site design for search engines. If you don't understand some of the following items, then you may want to discuss those items with your Web site developer. 1. Add educational Web pages. Educational Web pages are intended to educate the target audience about a particular topic (e.g., ICSI, assisted hatching, etc.), rather than to promote an organization's services or products. In addition to the fact that users like to read educational material, educational pages can dramatically help your Web site get more traffic from search engines for the following reasons: •
When your site has educational pages, other infertility Web site owners will be more likely to add a link pointer to your Web pages. And, the more Web pages that point to your Web pages, the higher search engines will rate your site popularity (called "link popularity"). The higher your Web site popularity, the higher your Web pages will appear in the search engine
• Many search engines index every Web page on a Web site. Thus, the more pages you have on your Web site, the more pages the search engines can index and the more likely that one of your pages will display as the result of a user's search. There is an additional reason why educational Web pages will increase the traffic to your Web site: •
You will gain the additional traffic directly from the Web sites that add links to your Web site. In particular, infertility directories (e.g., Infertility Resources2 or Infertility Professionals.com3) may want to add hyperlink pointers to your educational Web pages.
2. Do not build your Web site with frames. Background: Some Web site developers like to build Web sites using frames. A frame-based Web page is a special kind of page that divides the browser window into different areas called frames. Each frame area can display a different Web page. Although many Web site developers think it looks attractive and hip to use frames, there is a
significant downside to using frames. Using frames will cause immense difficulties in promoting your Web site. To assess whether your site is built in frames, check for the following conditions: •
Click on a few pages and look at the Web address in the location bar. If the Web address does not change, then it is probably a frame-based site.
•
Navigate to different Web pages. If there is one stationary block of the Web page that does not change, then it may be a frame-based Web site.
•
If you know how to do this while viewing the page in your Web browser, you can do a "File/View Source" to view the HTML. If you see references to "frames" in the HTML tags, then it is probably a framesbased site.
The problems: A "frame-based" Web site creates two major problems for promoting the site: •
Many search engines have trouble following the links in framebased Web sites.
•
Web directories cannot easily point to specific Web pages. They can only point to the frame-based site's home page. Continued on Page 21
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The Embr yologists’ Newsletter
Winter 2000
Continued from Page 20
Yet, the educational pages will be buried inside the site. In both cases, the result is that you will receive less traffic to your Web site. A frame-based Web site can be a major obstacle to getting lots of traffic from search engines and Web directories. Some Solutions: •
•
•
If you have not yet built your Web site, then build it using tables. Table-based pages allow for the same structured page design as frames. For example, all pages might have the same elements, including a top bar with your logo, a navigation bar and a bottom bar with copyright information. If your Web site is already built in frames, you can turn the frame-based pages into tablebased pages. If you or your Web page developer are unsure of how to do this, feel free to contact Internet Health Resources.1 You can also have a mixedmode site. The pages that you want indexed by the search engines can be table-based, and all other pages can be framebased.
For additional information about problems and solutions with frames, refer to "Build Your Web Site Without Frames."4 3. Ensure that the important keywords are in the Web page text, rather than inside the images. Background: Search engines regularly index millions of Web pages and keep track of the main keywords on those pages. If, for exam-
ple, an Internet user enters the word "infertility" into the search engine, it will query its database of keywords and return Web pages that contain the word "infertility."
a "navigation bar," which allows users to quickly access lots of pages on the Web site. The navigation bar hyperlinks can be either: •
Simple text link - this type of link is simple text, like the words on this Web page. You can assess whether a link is simple text by doing the following: If you can select and highlight the link with your cursor, then it's a simple text link. To do this, press your left mouse button and drag the cursor over the link to select the link. If you can highlight the link, it is a text link.
•
Image link - this type of link is an image that is made in a graphics program. It is not simple text; it is an image. The link is probably an image link if the following are true:
The problem: Search engines cannot "read" words inside of images and thus they won't index those words. If they don't index those words (e.g. "infertility” or "ICSI") then, when a person enters those words into a search engine, that Web page will not be found. Here are a couple of specific instances where this can be a problem: •
•
Some Web sites have a large image, with important words, on the home page. Some Web sites have major keywords (e.g., a list of services) contained inside of the images on the sub pages (e.g., the services page).
•
the link looks like an image (e.g., it is 3D or the font is very different from typical fonts on a page)
•
you cannot select and highlight the text with your cursor
Some Solutions: •
•
If you have an image that contains important keywords (e.g. a list of services) on your subpages, make sure that the words are also written in normal text. If you have a home page image with important text, consider taking the image apart as follows (a) keep the photo or line art as an image, and (b) turn the text inside the image into simple text.
4. Ensure that your major navigational links are text links. Background: The vast majority of Web pages have hyperlinks for accessing another Web page. Often the hyperlinks are grouped together on the top or side of the page into
The problem: Search engines index Web sites by first indexing the home page and then following the site links to other pages to index those pages. However, some search engines cannot follow image links. Consequently, if your navigation bar is made up of image links, those search engines will not be able to index many pages on your Web site. If they cannot index the pages, then they cannot add them to the search engine listing. Some solutions: In order to provide the text links for the search engines, you can do one of the following: Continued on Page 22
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The Embr yologists’ Newsletter
Winter 2000
Continued from Page 21
•
Keep your image bar with links and simply add the text links somewhere on the page (e.g., the bottom of the page).
•
Add a site map page that contains text links to all your sub pages.
•
Change the image links into simple text links.
5. Avoid using the exact same color for the text and the Web page background. Background: Some Web pages have text that is the same color as the Web page background. In this situation, it might seem that a user could not read text that is the same color (e.g., white) as the Web page background. However, consider a Web site with pages that have a white background and the following: •
a blue navigation bar with white hyperlink text or
•
a dark colored box (i.e., a table) with white text
However, search engines have caught onto this trick, and they now penalize people who attempt to use keyword spamming by (a) penalizing a Web page that has too many of the same keywords (e.g., "infertility" 100 times) on the page, and (b) some search engines penalize text that is the same color as the background under the assumption that the Web site owner is trying to spam the search engine. A solution: The basic solution is to change the color of the text to a color different from the page background. For example, you can change white text to a slightly off white color.
1. Internet Health Resources http://www.ihr.com 2. Infertility Resources http://www.ihr.com/infertility/ 3. InfertilityProfessionals.com http:// www.InfertilityProfessionals.com 4. "Build Your Web Site Without Frames" article http:// www.InfertilityWebsites.com/ development/ do_not_use_frames.html 5. Introduction to Search Engines http:// www.kcpl.lib.mo.us/search/ srchengines.htm
ADDITIONAL READING The following Web sites, not previously mentioned in this article, can provide additional information about ensuring that your Web site design is optimized for search engines: •
The problem: In the past, some people tried to fool the search engines by hiding keywords (e.g., "infertility") in the pages by making the keyword text the same color as the page background. That way normal users would not see, for example, the word "infertility" repeated 100 times. But the hope was that the "dumb" search engines would read the word and thereby think that this was a really important infertility page. This trick was called “keyword spamming.”
WEB SITE REFERENCES
•
Introduction to Search Engines5 Search Engine Watch home page6
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6. Search Engine Watch http:// www.searchenginewatch.com
❖ Cliff Bernstein, Ph.D., the author of this article, is the owner of Internet Health Resources (http://www.ihr.com), a company that builds and promotes infertility Web sites.