| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
,
,||,¶
From the Departments of Urology,
Biomedical Engineering,
Macromolecular Science and Engineering, ||
Obstetrics and Gynecology, and ¶
Molecular and Integrated Physiology, University
of Michigan, Ann Arbor, Michigan.
| Correspondence to: Gary D. Smith, 6428 Medical Sciences I Building, 1301 E Catherine Street, Ann Arbor, MI 48109-0617 (e-mail: smithgd{at}umich.edu) or Shuichi Takayama (e-mail: takayama{at}umich.edu). |
| Received for publication July 8, 2005; accepted for publication July 8, 2005. |
A promising new technology, microfluidics, exists and is becoming increasingly studied. This technology shows promise as an alternative for each step in the IVF process. Microfluidics, based on physical principles of fluid behavior in a microenvironment, has been used widely in chemistry and molecular biology applications (Tomlinson et al, 1995). Currently, microfluidics is gaining interest in studies of cellular behavior and interactions (Shim et al, 2003). In this article, we introduce basics of fluid behavior at the microscale and highlight previous uses of this technology outside of the reproductive sciences. We then describe fabrication of devices and review initial studies that used microfluidics in sperm sorting and microinsemination. Last, we point out some limitations of this new technology and provide speculation on future directions and application of microfluidics in ART.
Microenvironment Fluid Behavior
Fluid mechanics is a complex physical and mathematical science; therefore,
an extensive technical description and review of fluid physics is beyond the
scope and intent of this review. Instead, basic principles will be discussed
that govern fluid behavior in a microenvironment, especially those aspects
with a specific link to devices and technology currently being developed for
IVF. We have purposely avoided including mathematical details, choosing
instead to convey a general conceptual sense of fluid mechanics present within
microchannels. A comprehensive technical and mathematical description of
microfluidic physics can be found in excellent reviews from Beebe et al
(2002a) and Brody et al
(1996).
Fluids at the microscale are subject to forces typically not important at scales present in our everyday lives. Fluid at the scale of our normal environment is turbulent; particles within a stream of fluid move in an unpredictable pattern. Turbulent flow depends on certain fluid characteristics (viscosity, density, and velocity) and the geometry and size of the channel, leading to calculation of a value known as the Reynold's number. As the scale of the channel reaches micrometer levels, the Reynold's number decreases and becomes increasingly dependent on fluid characteristics. Decrease of the Reynold's number below a threshold value leads to fluid flow in a laminar fashion. Simply put, flow within microchannels becomes streamlined and predictable (Figure 1). At the microscale, fluid behavior becomes increasingly governed by viscous forces and surface tension, which can be described as the cohesiveness of the liquid's molecules.
|
This dominance by viscous forces results in several interesting phenomena. Flows with a low Reynold's number possess little to no momentum; thus, fluids within a microchannel respond quickly and reliably to changes in external forces. In addition, at the microscale, 2 or more streams of laminar flow in contact with each other do not mix, except by diffusion of molecules across the interface of the streams. The rate of diffusion between the contacting surfaces at the microscale can be very quick, partially because of the relatively short distances needed to cross fluid volumes.
Many of these fluid characteristics at the microscale form the principles driving the interest in the use of microchannels for gamete and embryo manipulation. In general, a microenvironment more closely resembles the in vivo conditions of fertilization and development when compared with a culture dish or drop of media. Below, we discuss the theory behind investigating the use of microfluidics in andrology, its testing, limitation, and potential future influence.
Microfluidics and Nonreproductive Cell Biology
Interest in microfluidics began with attempts to miniaturize chemical and
biological analysis devices in the laboratory
(Kricka, 1998). Current
designs are often referred to as "laboratory-on-a-chip" or
micrototal analysis systems (µTAS) and function by allowing a variety of
chemical processes and interactions to occur as fluid flows within their
miniature channels and chambers (Weigl and
Yager, 1999). Such devices perform all the analytical functions
necessary for their purpose, including sample handling, mixing, incubation,
sorting, transport, interaction, and detection or signaling within an
integrated microfluidic "chip." Examples include, but are not
limited to, immunoassays for antibodies present in serum
(Linder et al, 2002) and
assays determining enzyme reaction kinetics
(Xue et al, 2001;
Yakovleva et al, 2002).
Additional applications in cellular biology have emerged, such as integrated cell sorting devices working at the microscale (Fu et al, 2002) and microfluidic devices that allow for the study of cellular interactions with substrates or other cells (Shim et al, 2003). Advances in cell biology have been demonstrated with the use of microfluidics and the principle of laminar flow, allowing for selective exposure of subcellular areas of interest to membrane-permeable molecules (Takayama et al, 2001). Such precise delivery of molecules to cellular subdomains illustrates the precision with which microfluidic regulation of fluid flow is capable.
Advantages of such laboratory-on-a-chip technology are multiple. First, once designed and tested, the manufacture of such devices is straightforward and inexpensive, allowing them to be disposable (McDonald et al, 2000). Microfluidic analysis devices use very low volumes of samples and reagents and provide for faster reactions and response times (Weigl and Yager, 1999). Miniaturization very importantly allows for integration of multiple processes within a small, self-contained unit (Kricka, 1998). This can be translated into either multiple parallel analyses, consecutive serial processes, or both.
The brief overview given here is only intended to familiarize readers with the variety of capabilities of microfluidic technology and is by no means a comprehensive listing of microfluidic applications in sciences. Readers are encouraged to consult more thorough reviews (Khandurina and Guttman, 2002; Verpoorte, 2002).
Fabrication of Microfluidic Devices
Microfluidics systems were initially fabricated with the use of materials
and techniques common in the industry that inspired
them—microelectronics (McDonald et
al, 2000). Photolithography and etching of silicon and glass was a
highly developed technology also readily available to researchers interested
in miniaturizing analytical systems, yet costs were a significant barrier. In
search of a suitable alternative, polymers have quickly emerged as a material
for microfluidic biological device fabrication
(McDonald et al, 2000).
Compounds such as poly(methyl)methacrylate
(Martynova et al, 1997),
fluorinated ethylene propylene (Sahlin et
al, 2002), and poly(dimethylsiloxane) (PDMS;
McDonald and Whitesides, 2002) are cheaper and easier to manipulate than silicon-glass alternatives
(Martynova et al, 1997). PDMS
in particular has become one of the most actively explored and promising
materials thus far, possessing numerous characteristics specifically suitable
for biological use. It is nontoxic, transparent, insulating, and permeable to
gases (McDonald and Whitesides,
2002). From a fabrication standpoint, PDMS permits submicron
fidelity with molding, cures at low temperatures, and can easily seal
reversibly to itself and a host of other materials
(McDonald et al, 2000).
Although PDMS is generally regarded as nontoxic, special consideration must be given to its use with gametes and embryos, which can be very sensitive to their environment compared with transformed cell lines. Before the use of microfluidic devices with sperm, testing confirmed that no negative effects resulted from prolonged exposure to the materials used in their fabrication. Schuster et al (2003) reported that 30 minutes of exposure to PDMS did not alter sperm survival. In addition, Glasgow et al (2001) found that development of 2-cell mouse embryos to the blastocyst stage was unchanged by continuous exposure to numerous photolithography compounds compared with controls. Thus it appears that PDMS-composed microchannels or the materials used in their construction do not confer deleterious effects to gametes or embryos.
Microfluidics and Sperm Isolation
Numerous efforts have improved methods of semen processing and sperm
isolation. Currently, swim-up techniques or density gradient separation are
methods of choice (Trounson and Gardner,
2000). Both methods result in adequate recovery of motile sperm,
although additional steps might be necessary in poor-quality semen samples
(Bourne et al,
1995a,b).
However, some researchers have stated concern that these methods could
contribute to sperm morphological damage, DNA damage, production of
oxygen-free radicals, or multiple injuries
(Aitken and Clarkson, 1988;
Zini et al, 1999). In
addition, these techniques can be labor and time intensive. Ideal sperm
isolation would involve a simple, rapid, and atraumatic method to obtain
sufficient motile sperm for use in either IVF or ICSI, depending on need and
the quality of the original semen sample.
Attempts have been made to develop devices for such a purpose. The Wang tube (Wang et al, 1992), a uniquely configured glass tube, allows motile sperm to progress to an upper arm that is then separated for sperm use in intrauterine insemination or IVF. Comparison testing with swim-up and density gradient separation for normozoospermic samples revealed greater motility and morphology with the device (Wang, 1995). Lih et al (1996) have developed and tested a Lucite microchamber consisting of a central loading well surrounded by slightly depressed sidewells that was conceived from the observation that motile sperm migrate to the periphery of microdrops. This device concentrated motile sperm up to 13-fold in the sidewells, yielding a sufficient number for use in ICSI.
A microfluidic device has been explored for sperm diagnostic purposes. Kricka et al (1993) designed and fabricated silicon and glass devices for sperm motility evaluation. They evaluated sperm progression along the length of a microchannel (80 µm wide by 20 µm deep) and navigation through a network of branching channels. In initial studies, they demonstrated feasibility and hypothesized that this device could replace conventional methods of motility assessment and semen analysis. Subsequently, they demonstrated that sperm movement within microchannels, judged by the time needed to reach the end of the channel, correlated with forward progression scores (Kricka et al, 1997). However, the design of the device did not give reliable information regarding sperm concentration or percent motility and therefore could only serve as an adjunctive test of motility and forward progression rather than a comprehensive semen analysis tool.
Schuster et al (2003) developed a microfluidic device taking advantage of parallel laminar flow streams present at the microscale. In this device, a flowing stream of semen was placed in parallel with a flowing stream of media within a microchannel. Flow within microchannels was maintained by a novel gravity-driven, horizontally oriented pumping system developed specifically for the device (Cho et al, 2003). As discussed, these 2 parallel laminar flow streams mix only by diffusion. Motile sperm demonstrated the ability to actively propel themselves across contacting surface areas and deviated from the initial streamline into the media stream for collection, whereas nonmotile sperm and cellular debris remained in the initial stream and exited the device (Figure 2).
|
Testing of this laminar flow sorting system was performed with 40 µL of unprocessed human semen, followed by semen samples artificially filled with debris from a stock solution of round immature germ and white blood cells to simulate poor-quality samples. For unprocessed semen, the device consistently produced a sorted fraction with increased motility (mean 98% motile) and improved Kruger strict sperm morphology (mean 22% normal forms) compared with the initial specimen (mean 44% and 10%, respectively). For debris-filled samples, the device not only concentrated motile sperm (mean 98% motile) within the collected fraction, but was also able to produce a round cell:sperm ratio of 1:33 compared with a 10:1 ratio in the starting specimen (Schuster et al, 2003).
Microinsemination
Microfluidics might be particularly suitable for IVF for a number of
reasons (Suh et al, 2003). The
microenvironment of a microchannel more closely resembles in vivo
fertilization conditions than a culture dish or microdrop. Microfluidic
channels allow for nonturbulent bathing of gametes with fresh media throughout
insemination and coincubation. Sperm-oocyte interactions occur in an active
environment, rather than the static conditions present in a culture dish or
droplet. In addition, sperm can be predictably delivered via laminar flow to
each oocyte within the microchannel, eliminating the randomness of
sperm-oocyte interaction. In a culture dish, sperm can travel randomly in any
direction, thereby relying on random sperm movement toward the oocytes;
however, in a microchannel environment, sperm movement is limited by the
direction of flow, allowing for active transport to the oocytes. Finally,
microchannel environments use extremely small volumes of media, theoretically
requiring fewer sperm to achieve insemination concentrations equal to standard
IVF with larger volumes.
Previous investigators have attempted, with some success, to reduce the volume of insemination medium with various low-volume vessels, although none have gained widespread acceptance. Van der Ven et al (1989) tested the use of sterile, nonheparinized hematocrit capillary tubes (75 mm length, 0.9 mm inner diameter) for IVF in humans. Normospermic samples were used with standard culture tubes as controls. Volumes of 5-10 µL containing a range of 500-4000 sperm per oocyte were used in these capillary tubes. Overall fertilization rates between controls and capillary tubes was similar (78% and 66%, respectively), although slightly lower for sperm totals of 500-1000 (56%) compared with 2000-4000 total sperm (79%). Ranoux and Seibel (1990) used embryo cryopreservation straws in volumes up to 200 µL (Ranoux et al, 1988) with 2000-4000 motile sperm. Results compared favorably with controls, with 167 of 322 oocytes (51.8%) fertilized by the straw technique.
We have recently demonstrated that mouse IVF can be conducted successfully within microfluidic channels (unpublished data). Not only are lower total numbers of sperm required because of the use of reduced media volumes, we have also demonstrated murine fertilization within microchannels with lower insemination concentrations, further decreasing sperm requirements. We continue to develop design improvements that will result in increased efficiency and ease of use. Such microfluidic devices should ultimately be useful in clinical IVF, not only for oligospermic patients but potentially as a replacement for standard insemination.
Limitations and Future Applications
This microfluidic sperm sorting device provided a simple, atraumatic method
of obtaining motile sperm of normal morphology from both unprocessed normal
semen and poor-quality specimens containing significant debris. A limitation
of the device regards the flow rate, estimated at 
20-40 µL/h. In its
current form, it is not capable of processing an entire semen specimen;
however, it does provide a means of quickly and easily isolating a small
sample of motile sperm of normal morphology for ICSI, insemination in
microdrops under oil, or microinsemination in an integrated microfluidic
device (Clark et al, 2003;
unpublished data). In addition, modifications and improvements in the design
are in progress that might allow for large-scale processing in parallel and
increased efficiency of flow and sorting
(Schuster et al, 2003).
As with any new technology, design plays an important role. Improvements in loading methods, which can allow for visualization under magnification without adjustment of the microscope focus, should improve outcomes. Addition of oocytes and sperm to the device under a microscope, which requires time outside of the humidified 5% CO2 environment, has significant deleterious effects on gamete health and survival. Reduction of this time currently requires a significant learning curve. Last, the implementation of more sophisticated but less operator-reliant mechanisms for fluid flow should improve efficiency. Current studies are focused on a variation of the gravity-driven, horizontally oriented reservoir pumping system (Zhu et al, 2004) from the microfluidic sperm sorter developed by Cho et al (2003) and its application to microfluidic insemination.
Although much of the work with microfluidics in IVF has been performed in a stepwise fashion, the ultimate goal of process miniaturization and microfluidic technology is integration. Use of microfluidic technology for sperm processing ultimately results in a small volume and fraction of motile sperm. Such volumes are difficult to subsequently use and translate into a macroscale environment. However, laminar flow-sorted sperm have been used for subsequent fertilization within a microenvironment (unpublished data). Integration of a microfluidic channel for the oocyte and the collection stream of sorted sperm would result in automatic coincubation of the oocyte with these motile sperm. Following insemination, the oocyte can be directed to a secondary site for cumulus removal, evaluation for fertilization, and embryo culture (see review in Beebe et al, 2002b). Sequential media can be provided for ideal embryo development. Each step logically follows the other, with no cell manipulation other than directing flow along a variety of channels. Miniaturization allows the entire system to be small and self-contained. Decreased intervention by laboratory personnel not only decreases gamete and embryo manipulation but also provides for greater consistency of incubation conditions.
|
Footnotes
* Andrology Lab Corner welcomes the submission of unsolicited
manuscripts, requested reviews, and articles in a debate format. Manuscripts
will be reviewed and edited by the Section Editor. All submissions should be
sent to the Journal of Andrology Editorial Office. Letters to
the editor in response to articles as well as suggested topics for future
issues are encouraged. 
References
Aitken RJ, Clarkson JS. Significance of reactive oxygen species and
antioxidants in defining the efficacy of sperm preparation techniques.
J Androl. 1988;9: 367
-376.
Beebe DJ, Mensing GA, Walker GM. Physics and applications of microfluidics in biology. Ann Rev Biomed Eng. 2002a; 4: 261 -286.[CrossRef][Medline]
Beebe DJ, Wheeler M, Zeringue H, Walters E, Raty S. Microfluidic technology for assisted reproduction. Theriogenology. 2002b; 57: 125 -135.[CrossRef][Medline]
Bonduelle M, Camus M, De Vos A, Staessen C, Tournaye H, Van Assche E, Verheyen G, Devroey P, Liebaers I, Van Steirteghem A. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod. 1999; 14(suppl 1): 243 -264.
Bourne H, Richings N, Harari O, Watkins W, Speirs AL, Johnston WI, Baker HW. The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility. Reprod Fertil Devel. 1995a;7: 237 -245.[CrossRef][Medline]
Bourne H, Richings N, Liu DY, Clarke GN, Harari O, Baker HW. Sperm preparation for intracytoplasmic injection: methods and relationship to fertilization results. Reprod Fertil Devel. 1995b; 7: 177 -183.[CrossRef][Medline]
Brody JP, Yager P, Goldstein RE, Austin RH. Biotechnology at low Reynolds numbers. Biophys J. 1996; 71: 3430 -3441.[Medline]
Cho B, Schuster TG, Zhu X, Chang D, Smith GD, Takayama S. Passively driven integrated microfluidic system for separation of motile sperm. Anal Chem. 2003; 75: 1671 -1675.[Medline]
Clark SG, Walters E, Beebe DJ, Wheeler MB. A novel integrated in vitro maturation and in vitro fertilization system for swine. Theriogenology. 2003; 59: 441 .
Davis JA, Raty S, Eddington DT, Glasgow IK, Zeringue HC, Wheeler MB, Beebe DJ. Development of microfluidic channels for the culture of mammalian embryos. In: 1st Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology. Lyon, France: 2000; 307-310.
Fu AY, Chou HP, Spence C, Arnold FH, Quake SR. An integrated microfabricated cell sorter. Anal Chem. 2002; 74: 2451 -2457.[Medline]
Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod. 1998; 13: 148 -159.
Glasgow IK, Zeringue HC, Beebe DJ, Choi SJ, Lyman JT, Chan NG, Wheeler MB. Handling individual mammalian embryos using microfluidics. IEEE Trans Biomed Eng. 2001; 48: 570 -578.[CrossRef][Medline]
Gu W, Zhu X, Futai N, Cho BS, Takayama S. Computerized microfluidic
cell culture using elastomeric channels and Braille display. Proc
Natl Acad Sci U S A. 2004; 101: 15861
-15866.
Khandurina J, Guttman A. Bioanalysis in microfluidic devices. J Chromatogr A. 2002; 943: 159 -183.[CrossRef][Medline]
Kricka LJ. Miniaturization of analytical systems. Clin
Chem. 1998;44: 2008
-2014.
Kricka LJ, Faro I, Heyner S, Garside WT, Fitzpatrick G, McKinnon G, Ho J, Wilding P. Micromachined analytical devices: microchips for semen testing. J Pharm Biomed Anal. 1997; 15: 1443 -1447.[CrossRef][Medline]
Kricka LJ, Nozaki O, Heyner S, Garside WT, Wilding P. Applications
of a microfabricated device for evaluating sperm function. Clin
Chem. 1993;39: 1944
-1947.
Lih CH, Obasaju M, McCaffrey C, Gordon JW. Development of a microchamber which spontaneously selects high-quality sperm for use in in vitro fertilization or micromanipulation. J Assist Reprod. Genet. 1996;13: 657 -662.[CrossRef][Medline]
Linder V, Verpoorte E, de Rooij NF, Sigrist H, Thormann W. Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard. Electrophoresis. 2002; 23: 740 -749.[CrossRef][Medline]
Martynova L, Locascio LE, Gaitan M, Kramer GW, Christensen RG, MacCrehan WA. Fabrication of plastic microfluid channels by imprinting methods. Anal Chem. 1997; 69: 4783 -4789.[Medline]
McDonald JC, Duffy DC, Anderson JR, Chiu DT, Wu H, Schueller OJ, Whitesides GM. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis. 2000; 21: 27 -40.[CrossRef][Medline]
McDonald JC, Whitesides GM. Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc Chem Res. 2002; 35: 491 -499.[CrossRef][Medline]
Mortimer D. Practical Laboratory Andrology. New York, NY: Oxford University Press; 1994.
Pool TB. Recent advances in the production of viable human embryos in vitro. Reprod Biomed Online. 2002; 4: 294 -302.[Medline]
Ranoux C, Poirot C, Foulot H, Dubuisson JB, Aubriot FX, Chevallier O, Cardone V. Human egg fertilization in capillary tubes. J In Vitro Fertil Embryo Transfer. 1988; 5: 49 -50.[CrossRef][Medline]
Ranoux C, Seibel MM. New techniques in fertilization: intravaginal culture and microvolume straw. J In Vitro Fertil Embryo Transfer. 1990;7: 6 -8.[CrossRef][Medline]
Sahlin E, Beisler AT, Woltman SJ, Weber SG. Fabrication of microchannel structures in fluorinated ethylene propylene. Anal Chem. 2002;74: 4566 -4569.[Medline]
Schuster TG, Cho B, Keller LM, Takayama S, Smith GD. Isolation of motile sperm from semen samples using microfluidics. Reprod Biomed Online. 2003;7: 75 -81.[Medline]
Shim J, Bersano-Begey TF, Zhu X, Tkaczyk AH, Linderman JJ, Takayama S. Micro- and nanotechnologies for studying cellular function. Curr Topics Med Chem. 2003;3: 687 -703.
Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet. 1978; 2: 366 .
Suh RS, Phadke N, Ohl DA, Takayama S, Smith GD. Rethinking
gamete/embryo isolation and culture with microfluidics. Hum Reprod
Update. 2003;9: 451
-61.
Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE, Whitesides GM. Subcellular positioning of small molecules. Nature. 2001; 411: 1016 .[CrossRef][Medline]
Tomlinson AJ, Guzman NA, Naylor S. Enhancement of concentration limits of detection in CE and CE-MS: a review of on-line sample extraction, cleanup, analyte preconcentration, and microreactor technology. J Capillary Electrophoresis 1995; 2: 247 -266.
Trounson AO, Gardner DK. Handbook of In Vitro Fertilization. 2nd ed. Boca Raton, Fla: CRC Press Press LLC; 2000 .
van der Ven HH, Hoebbel K, Al Hasani S, Diedrich K, Krebs D.
Fertilization of human oocytes in capillary tubes with very small numbers of
spermatozoa. Hum Reprod. 1989; 4: 72
-76.
Verpoorte E. Microfluidic chips for clinical and forensic analysis. Electrophoresis. 2002; 23: 677 -712.[CrossRef][Medline]
Wang FN. Real-time sperm separation system: a review of Wang tubes and related technologies. Arch Androl. 1995; 34: 13 -32.[Medline]
Wang FN, Lin CT, Hong CY, Hsiung CH, Su TP, Tsai HD. Modification of the Wang tube to improve in vitro semen manipulation. Arch Androl. 1992;29: 267 -269.[Medline]
Weigl BH, Yager P. Microfluidic diffusion-based separation and
detection. Science. 1999; 283: 346
-347.
Xue Q, Wainright A, Gangakhedkar S, Gibbons I. Multiplexed enzyme assays in capillary electrophoretic single-use microfluidic devices. Electrophoresis. 2001; 22: 4000 -4007.[CrossRef][Medline]
Yakovleva J, Davidsson R, Lobanova A, Bengtsson M, Eremin S, Laurell T, Emneus J. Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection. Anal Chem. 2002;74: 2994 -3004.[Medline]
Zhu X, Chu LY, Chueh B-H, Shen M, Hazarika B, Phadke N, Takayama S. Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation. Analyst. 2004;129: 1026 -1031[CrossRef][Medline]
Zini A, Mak V, Phang D, Jarvi K. Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity. Fertil Steril. 1999;72: 496 -499.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
