Automated sperm concentration analysis with a new flow cytometry-based device, S-FCM.
TL;DR: Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses, showing that automated analyses were highly reproducible and the Carryover rate was 0.17% or less.
Abstract: The S-FCM uses flow cytometry technology to measure sperm concentrations. Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses. In addition, sperm concentrations measured with the S-FCM were compared with those obtained by manual analyses with the Makler chamber and the improved Neubauer hemacytometer. The results showed that automated analyses with the S-FCM were highly reproducible and the carryover rate was 0.17% or less. In within-run imprecision assays, the coefficients of variation for the S-FCM were less than 5% at all sperm concentrations, while those for the Makler chamber were between 17.7% and 28.7% at lower sperm concentrations. The overall correlation between values measured with the S-FCM and those measured with the Makler chamber and improved Neubauer hemacytometer was excellent, but at lower sperm concentrations the correlation was lower. The S-FCM performed sperm concentration analyses in 110 seconds compared with 5 minutes for the Makler chamber and 10 minutes for the improved Neubauer hemacytometer. The S-FCM is suitable for quantitative measurement of lower sperm concentrations. Sperm counts are an essential step in the evaluation of male fertility and usually are performed by microscopic examination of semen by trained personnel. The World Health Organization (WHO) recommends the use of a hemacytometer for determining sperm concentrations in semen, 1 and several methods of using these devices have been described. 1-3 However, the accuracy and reproducibility of analytic data obtained with hemacytometers reportedly are not good enough to compare results from different institutions. 4-6 A number of studies have shown that manual counting is associated with the greatest potential for gross analytic errors in measuring sperm concentrations. 4-8 Consequently, computer-assisted semen analyzers were developed to overcome the subjectivity of sperm counting, but most of these analyzers use some sort of counting chamber or hemacytometer that is itself a source of intrinsic errors. When using a counting chamber or hemacytometer, only spermatozoa located in it can be counted, and only very small quantities of specimens are used to estimate the sperm concentration in 1 mL of semen. Recently we developed a new device (S-FCM) for measuring sperm concentrations that uses flow cytometry technology and an argon laser as the light source. To improve accuracy, the S-FCM device uses 90 times greater quantities of semen than are used with a counting chamber or hemacytometer. In this study, we compared the accuracy and reproducibility of the S-FCM with that of the Makler sperm counting chamber. We also evaluated the feasibility of using the S-FCM in clinical settings by comparing the analytic data with those obtained by using the improved Neubauer hemacytometer or the Makler chamber.
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TL;DR: Disposable chambers are suitable for routine semen analysis with CASA in a diagnostic andrology setting with the proper workflow and quality control and a plain glass slide and a tissue culture dish cover used with a coverslip showed rather better performance in semen assessment.
Abstract: A routine computer-assisted sperm analysis is an important diagnostic test in the andrology laboratory. To evaluate the accuracy and precision of the different types of counting chambers for human semen analysis in combination with a computer-assisted semen analyzer (CASA), a quality-control study that compared human sperm analysis results obtained using different counting chambers (Makler chamber, disposable 8-cell GoldCyto chamber, disposable 4-cell Leja chamber, a plain glass slide, and a tissue culture dish cover with a 24 × 24 mm2 coverslip) in conjunction with the CASA systems was performed. Significantly higher counts of sperm concentration were obtained from the reusable Makler chamber than from the other counting chambers. Sperm motility from drop loaded counting chambers was significantly higher than that of capillary-loaded chambers. A plain glass slide and a tissue culture dish cover used with a coverslip showed rather better performance in semen assessment. Disposable chambers are sui...
13 citations
Cites methods from "Automated sperm concentration analy..."
...Generally, the semen analysis is the routine first andrology laboratory test given in male infertility clinics [Tsuji et al. 2002]....
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...Generally, the semen analysis is the routine first andrology laboratory test given in male infertility clinics [Tsuji et al. 2002]....
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...A 5-mL drop of semen was placed on the loading area of a Makler chamber and the coverslip was applied immediately [Christensen et al. 2005; Matson et al. 1999; Tsuji et al. 2002]....
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Journal Article•
TL;DR: The study indicated that there was no significant difference in acrosomal integrity among the breeds and the values mostly correlates with the guideline of Minimum Standard Protocol for Production of bovine semen, 2012 of Govt.
Abstract: Acrosomal integrity and sperm concentration are two important parameters to assess the quality of frozen semen doses which in terms validates the fertilizing capacity and conception rate. The present study was undertaken to evaluate acrosomal integrity by Giemsa's stain and sperm concentration of FSS using improved neubauer chamber in Exotic pure Jersey, Crossbred Jersey, Indigenous Gir cattle and Indigenous Murrah buffalo prior to the field use. The overall values of Giemsa's stain were observed as 73.74±0.31, 18.65±0.33 and 7.79±0.25 percent for Intact Acrosome, Partially Damaged Acrosome and Fully Damaged Acrosome, respectively. Overall values of sperm concentration were 21.98±0.28 million per straw. The study indicated that there was no significant difference (P<0.05) among the breeds and the values mostly correlates with the guideline of Minimum Standard Protocol for Production of bovine semen, 2012 of Govt. of India.
12 citations
Cites background from "Automated sperm concentration analy..."
...flow cytometry (Takumi et al., 2002) and Computer Assisted Semen Analyzer (CASA) (Coetzee t al....
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...…sperm concentration -two vital... Exploratory Animal and Medical Research, Vol.4, Issue 1, June, 2014 sophisticated techniques i.e. flow cytometry (Takumi et al., 2002) and Computer Assisted Semen Analyzer (CASA) (Coetzee t al., 2001), these instruments also have problems in reproducibility and…...
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01 Jan 2011
TL;DR: This work has shown that cell dehydration in Cryopreservation affects the ability of sperm to be converted into Vitrified Sperm in a non-equilibrium manner and this has implications for fish conservation.
Abstract: .................................................................................................................................. xvii Chapter 1. Foreword ............................................................................................................... 1 Biomedical Research Fish Models..................................................................................... 3 Genetically Improved Lines ............................................................................................... 4 Endangered Fish Species ................................................................................................... 4 References .......................................................................................................................... 6 Chapter 2. Introduction ........................................................................................................... 11 Cell Dehydration in Cryopreservation ............................................................................... 11 Equilibrium vs. Non-equilibrium Cryopreservation .......................................................... 14 Source of Variation in Vitrification ................................................................................... 18 Advantages of Vitrification ................................................................................................ 18 Sperm Vitrification ............................................................................................................ 19 Vitrification of Fish Eggs and Embryos ............................................................................ 22 New Strategies for Application of Cell Vitrification ......................................................... 24 References .......................................................................................................................... 25 Chapter 3. Production of Channel Catfish with Sperm Cryopreserved by Rapid Non-Equilibrium Cooling ........................................................................... 37 Materials and Methods ....................................................................................................... 41 Results ................................................................................................................................ 50 Discussion .......................................................................................................................... 56 Conclusions ........................................................................................................................ 64 References .......................................................................................................................... 65 Chapter 4. Production of F1 Offspring with Vitrified Sperm from a Live-bearing Fish, the Green Swordtail Xiphophorus hellerii ............................................................. 75 Materials and Methods ....................................................................................................... 77 Results ................................................................................................................................ 84 Discussion .......................................................................................................................... 91 References .......................................................................................................................... 101
8 citations
TL;DR: Different methods for the estimation of sperm concentrations like hemocytometry, spectrophotometry, microcells, plate reader, image analysis and finally flow cytometry are compared and contrasted and their relative merits and demerits are discussed with a detailed review of literature.
Abstract: Sperm count assessments form the essential component of the diagnostic and prognostic evaluation of male fertility, according to guidelines of WHO (1992). The problem of subjective bias, inter and intra operator variability of reporting is discussed in this paper. The problem of inter operator variability has been improved and reproducibility has been made more objective with the introduction of computer-assisted semen analysis (CASA) protocols. To overcome the stated limitations and achieve objective assessment with a high precision, a new technique called flow cytometry was developed. Different methods for the estimation of sperm concentrations like hemocytometry, spectrophotometry, microcells, plate reader, image analysis and finally flow cytometry are compared and contrasted. Their relative merits and demerits are discussed with a detailed review of literature. Methods for estimation of sperm concentration are discussed in this paper.
Key words: Sperm counts, semen analysis, flow cytometry.
7 citations
Cites background from "Automated sperm concentration analy..."
...All these had led to the increased use of flow cytometry to estimate sperm concentrations and to bring concurrent agreement between different laboratories (Eustache et al., 2001; Tsuji et al., 2002)....
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TL;DR: In this issue, Jin-Chin Lu and co-workers compare several methods for determining sperm concentration, including flow cytometry, computer-assisted sperm analysis and manual microscopic assessment.
Abstract: In this issue, Jin-Chin Lu and co-workers compare several methods for determining sperm concentration, including flow cytometry, computer-assisted sperm analysis and manual microscopic assessment [1]. Accurate determination of the sperm number in an ejaculate is important in the evaluation of infertile couples, in reproductive toxicology and in testing potential male contraceptives. Although the results of semen analysis cannot be taken as predictive of the fertility potential of a man, they may provide important clinical information. Assessment of sperm concentration is a central step in routine semen analysis, and studies have shown that sperm concentration and total sperm count are related to natural pregnancies [2–5]. Traditionally, sperm concentration has been determined by microscopic examination of the semen, and the method recommended by the World Health Organization (WHO) involves use of a haemocytometer [6]. Since preparation of the sample and counting 200 spermatozoa in duplicate are often considered laborious, and include several potential sources of error, efforts have been made worldwide to establish an accurate and rapid method for sperm counting. A variety of counting chambers have been developed, some of them intended for undiluted samples. Precision may be low with undiluted samples, however, since it is more challenging to enumerate a moving sperm than an immobilized one. Furthermore, in some chambers there is no uniform distribution of the spermatozoa, which means that assessment has to be done at several different distances from the site of loading [7]. Comparative studies have revealed considerable differences in accuracy, precision and agreement between counting chambers [8–12]. Systems for digital image semen analysis were developed in an attempt to obtain an objective measurement of semen characteristics, especially for sperm movements, and introduced in the 1980s [13]. In obtaining reproducible values for sperm concentration with this method it is important to use DNA-specific stain in order to distinguish
4 citations
References
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TL;DR: An external quality control study for semen analysis was performed involving 10 andrology laboratories in geographically separate locations and the coefficients of variation for sperm counts varied with sperm concentrations showing highest variability for low and lowest for high concentrations.
203 citations
200 citations
TL;DR: It is concluded that results of automated semen analysis may not be comparable among different laboratories unless identical parameter settings are used.
117 citations
TL;DR: Semen samples from 77 men were used to estimate the accuracy and precision of measurements of sperm density, percent motility, and motion characteristics using a new, fully automated, computer-assisted semen analyzer (CASA).
102 citations
TL;DR: Easy performance, rapid sperm counts, and improvement of motility estimation make this chamber a useful tool where sperm analysis is carried out.
97 citations