Integration of Micro Fluidic Bio-chip Design and Automatic fluorescent Identification for Rapid Sperm Mobility Assessment
01 Jan 2009-pp 332-335
TL;DR: The proposed method is cost effective and is possible to serve as a protocol for rapid sperm quantitative assessment and confirmed that live sperms were able to cross the laminar stream created by the device and reach the sorting chamber with corresponding range from non-motile sperm to debris, as well as from living to dead ones.
Abstract: Since male abnormality has now been reported to account at least thirty percent of couples with fertility problem. Therefore, distinguishing and classification of sperm has become more and more important, especially when Assisted Reproductive Technology (ART) like in vitro fertilization (IVF) is involved. However, traditional sperm sorting methods all requires manual selection, which is tedious and often cause sperm damage during the screen process. Although, new design from our recent study, seemed to point for a possibility to make these kind of MISS device capable of separating sperms into various mobility sub-class. In addition, the Hoechst 33258 and propidium iodide double staining results, also confirmed that the most motile and health sperm is indeed able to leave the laminar stream and being sorted out at outlet-h, with least dead sperm or debris. Meanwhile, we observed the dead sperm had been dominate from clear red stains and live sperm are not as active as is corresponded with more diffused blue stains. In order to confirm the real sorting situation, we counted the amount of sperm in collected sample by flow cytometry and tried to establish the statistic model. Though the flow cytometry, we could detect the sperm fluorescent distribution and count the size of sperm population. Nevertheless, t-test with 95% confidence level also shown significant difference in live/dead sperm ratio. Therefore, from the combined result among flow cytometry, fluorescent stain, and the statistic model test, it is confirmed that live sperms were able to cross the laminar stream created by the device and reach the sorting chamber with corresponding range from non-motile sperms to debris, as well as from living to dead ones. It is concluded that the proposed method is cost effective and is possible to serve as a protocol for rapid sperm quantitative assessment.
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TL;DR: The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI), a newly developed fluorescent nucleic acid stain that stained the nuclei ofliving sperm bright green as determined by simultaneous examination of fluorescence and motility.
Abstract: The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI). SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. Conversely, PI stained only nonmotile sperm that had lost their membrane integrity. Sperm from bulls, boars, rams, rabbits, mice, and men were stained and examined through use of fluorescence microscopy. The proportions of living and dead sperm were determined by first staining with SYBR-14 and PI and then assessing stain uptake by flow cytometry. Similar staining patterns were observed in all six mammalian species tested. Three populations of sperm were identified: living--SYBR-14 stained, dead--PI stained, and moribund--doubly stained. The SYBR-14 staining was replaced by PI staining as sperm progressed from living to moribund. The transition from green (SYBR-14) to red (PI) fluorescence started at the posterior region of the sperm head and proceeded anteriorly. The proportions of living and dead sperm in mammalian semen were readily identified through use of dual staining with SYBR-14 and PI and quantified through use of flow cytometry.
659 citations
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TL;DR: Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media and caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility.
Abstract: The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.
415 citations
"Integration of Micro Fluidic Bio-ch..." refers background in this paper
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TL;DR: A self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques, and opens the way for convenient bioassays based on sperm motility including at-home motiles sperm tests.
Abstract: This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.
295 citations
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TL;DR: There is a male factor in infertility in half of the couples, and even after a thorough evaluation, the cause of a man's lack of normal fertility usually remains unknown.
Abstract: Fifteen percent of couples are subfertile — that is, they have less-than-normal fertility. In approximately 30 percent of the cases, an important abnormality is identified in only the man, and in another 20 percent abnormalities are detected in both partners. Thus, there is a male factor in infertility in half of the couples. The evaluation should begin with a complete history taking, physical examination, and appropriate laboratory tests. Unfortunately, even after a thorough evaluation, the cause of a man's lack of normal fertility usually remains unknown. Since it is very difficult to develop a rational treatment plan to correct a . . .
132 citations
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TL;DR: A microfluidic system with embedded reservoirs that sorts sperm based on the ability of motile sperm to move out of their initial streamlines in a laminar fluid stream that pumps fluids by hydrostatic pressure and surface tension is described.
Abstract: In vitro fertilization using small amounts of cryopreserved sperm would benefit from efficient microscale methods to sort "healthy" motile sperm from nonmotile ones. This paper describes a microfluidic system with embedded reservoirs that sorts sperm based on the ability of motile sperm to move out of their initial streamlines in a laminar fluid stream. The device has horizontally oriented sample inlet and outlet reservoirs with different relative heights that serve the dual purpose of also being a pumping system that pumps fluids by hydrostatic pressure and surface tension. Sperm sorting is necessary for cryopreservation and intracytoplasmic sperm injection of sperm from men with low viable sperm counts. Since they have few viable sperm, it is difficult to obtain the sperm in an efficient manner using current procedures.
30 citations
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