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Andrology Lab Corner* |
From the * Reproductive Medicine Center,
University of Minnesota, Minneapolis, Minnesota;
Andrology Laboratory, Department of Obstetrics
and Gynecology, University of Rochester Medical Center, Rochester, New York;
Center for Reproductive Medicine of New
Mexico, Albuquerque, New Mexico; Department of
Obstetrics and Gynecology, Geisinger Medical Center, Danville, Pennsylvania;||
Office of Medical Research, Children's Mercy
Hospital, Kansas City, Missouri; and ¶ SAGE
BioPharma, San Clemente, California.
| Correspondence to: Christopher De Jonge PhD HCLD, Reproductive Medicine Center, 606 24th Avenue South, Suite 500, Minneapolis, MN 55454 (e-mail: dejonge{at}umn.edu). |
| Received for publication September 6, 2002; accepted for publication September 6, 2002. |
Rationale for Quality Control and Assurance
All products used in ART laboratories that directly or indirectly contact
gametes and embryos must have no negative influence on organism viability or
function (Parinaud et al,
1987). In addition, all products that directly contact gametes and
embryos must be tested for toxicity before clinical implementation. In order
for a laboratory to detect toxicity it must first have a reliable method for
making that determination. The most appropriate mechanism for quality
control/quality assurance (QC/QA) testing is to use a bioassay. The most
appropriate bioassay is one that provides a sensitive and robust monitoring
system that approximates the conditions that will be experienced by gametes
and embryos during their in vitro lifetime. To ensure that the method being
used performs optimally a laboratory should participate in an external
proficiency-testing program.
Current Methods for QC/QA
Three bioassays are in common use for determining the acceptability of
media and materials. The first and with perhaps the longest history of use is
the mouse embryo assay (Ackerman et al,
1984a,b).
This assay involves the culture of frozen or nonfrozen, 1- or 2-cell mouse
embryos to the blastocyst stage. The mouse embryo assay is easy to perform
simply by virtue of the commercial availability of biological materials.
However, the use of mouse embryos has several downsides, such as the reporting
system for toxicity (eg, Fleming et al,
1987; Scott et al,
1993), and different mouse strains vary in their sensitivity to
the same test conditions (Fleetham et al,
1993; Scott et al,
1993). Further, there is no apparent consensus on which mouse
embryonic cell stage (eg, 1- or 2-cell) is best for use in testing
(Davidson et al, 1988). There
are cost issues as well, related both to the purchase of embryos on a routine
basis and liquid nitrogen dewars for the storage of separate human cells and
tissues. Finally, animal rights issues may also be a concern with the use of
this assay.
The second most commonly used bioassay for QC and proficiency testing is the human sperm survival assay (Critchlow et al, 1989; Claassens et al, 2000). This assay is convenient to perform and materials are readily available. Further, in the context of an ART laboratory, the cell type in question is familiar to testing personnel and is compatible with the clinical laboratory environment.
A less commonly used but still relevant bioassay is the hamster sperm motility assay (Bavister and Andrews, 1988; Rinehart et al, 1988). This assay has a consensus in use of materials and methods alike. Similar to the mouse assay, the hamster assay can be somewhat expensive, cumbersome, and its use may also be offensive to animal rights groups.
The purpose of the present study was to determine whether the human sperm survival assay, based on the work by Claassens et al (2000), could be used effectively in a multicenter trial using a consensus protocol and whether it would yield consistent results between laboratories.
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Materials and Methods |
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 Top Materials and Methods ResultsConclusionsReferences |
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Sperm Processing
Aliquots of frozen donor semen (multiple specimens from each donor) and
in-house fresh specimens were tested. Fresh semen was allowed to liquefy and
cryopreserved semen was allowed to thaw prior to assessing semen parameters
following World Health Organization criteria
(WHO, 1999). Semen was layered
over a discontinuous, two-layer (47%/90% v:v) PureCeption (SAGE BioPharma, San
Clemente, Calif) density gradient and centrifuged at 300 x g
for 25 minutes. Sperm pellets were resuspended in 1 mL of Quinns Hepes human
tubal fluid (HTF) medium (SAGE BioPharma) containing 5 mg/mL of human serum
albumin (HSA) and centrifuged at 300 x g for 5 minutes. Sperm
pellets were resuspended in 37°C CO2-equilibrated
bicarbonate-buffered HTF (SAGE BioPharma) and sperm parameters were assessed.
Aliquots were removed and added to tubes containing HTF to achieve a final
motile sperm concentration of 5 x 106/mL. All media,
including HSA, were from SAGE BioPharma.
Incubation Conditions
Sperm were incubated (0.5 mL) at 37°C in a humidified 5.0%
CO2 incubator under the following conditions: 1) HTF, 2) HTF with 5
mg/mL HSA, 3) adulterated HTF, and 4) adulterated HTF with 5 mg/mL HSA. At 2,
4, 6, 8, 24, and 48 hours aliquots were removed and added to the same volume
of Hepes HTF for sperm motility assessment.
Statistical Analysis
Data were collated and analyzed using a mixed model ANOVA and linear
regression. Slopes from regression lines were used for comparison. The
percentage change from baseline (time = 0) was the dependent variable.
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Results |
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TopMaterials and MethodsResultsConclusionsReferences |
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Four laboratories had similar slope of motility patterns: control + HSA slope of motility > control - HSA slope of motility > treatment + HSA slope of motility > treatment - HSA slope of motility. The fifth laboratory had a slope of motility pattern as follows: control + HSA > treatment + HSA > control - HSA > treatment - HSA. The incubation conditions in laboratory 5 were not consistent (CO2 = 6.0) with the other four laboratories, and therefore the remaining data consist of only the four laboratories that used identical incubation conditions. It is important to indicate that for control and treatment conditions, when HSA was present the regression lines for laboratory 5 were comparable to the other four laboratories.
Table 1 shows the mean motility regression line slopes for the four laboratories. A negative regression line slope value reflects a decline in motility over the 48-hour incubation time. Precipitous declines in sperm motility over the 48-hour incubation time are reflected by elevated negative mean regression line slope values. When HSA was included in control and adulterated medium, the latter had a threefold steeper mean motility regression line slope compared to that of the control. When HSA was excluded from control and adulterated medium, the latter had a fivefold steeper mean motility regression line slope compared to that of the control. If the data for treatment without HSA from laboratory 1 are excluded, then the apparent difference in mean motility regression line slopes between media with or without HSA is negated.
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Table 2 reflects the mean regression values for the same donor tested at different laboratory locations. These data are consistent with those in Table 1, in which mean regression line slope values are greater (reflective of a more precipitous rate of decline in motility) for sperm incubated in adulterated medium compared with sperm incubated in control medium. In addition, there appears to be greater sensitivity to the adulterant, as indicated by the regression line slope values, when HSA is excluded from the medium. There is also an apparent greater variation in the rate at which sperm lost motility under these conditions as suggested by the large standard deviations.
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Conclusions |
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TopMaterials and MethodsResultsConclusionsReferences |
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Quality control and proficiency testing for ART laboratories has historically been problematic. The commonly used mouse embryo assay suffers from strain dependence, high cost, and other negative factors. As demonstrated by the results herein, the sperm survival assay, when performed under controlled conditions, can be used effectively as part of standard quality monitoring and proficiency testing programs. The inherent problems found with use of other bioassays currently in use are minimized or eliminated with the use of the human sperm survival assay.
The human sperm survival assay performs well as a bioassay for testing gamete toxicity by contact materials, reagents, and media, which are essential for ART laboratories. However, it is important to caution that the sensitivity of the sperm survival assay remains to be established. These results also show that performance data from the sperm survival assay between different laboratories is comparable, and thus can be not only compared, but used in proficiency testing regimens, and other inter-laboratory tests of competence.
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Footnotes |
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References |
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TopMaterials and MethodsResultsConclusionsReferences |
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Ackerman SB, Taylor SP, Swanson RJ, Laurell LH. Mouse embryo culture for screening in human IVF. Arch Androl.1984b; 12(suppl):129 -136.
Bavister BD, Andrews JC. A rapid sperm motility bioassay procedure for quality-control testing of water and culture media. J IVFET. 1988;5:67 -75.
Carrell DT, Cartmill D. A brief review of current and proposed
federal government regulation of assisted reproduction laboratories in the
United States. J Androl.2002; 23:611
-617.
Claassens OE, Wehr JB, Harrison KL. Optimizing sensitivity of the
human sperm motility assay for embryo toxicity testing. Hum
Reprod. 2000;15:1586
-1591.
Clinical Laboratory Improvement Amendments of 1988: Final Rule, 57Federal Register 7002 (1992).
Critchlow JD, Matson PL, Newman MC, Horne G, Troup SA, Lieberman
BA. Quality control in an in-vitro fertilization laboratory: use of human
sperm survival studies. Hum Reprod.1989; 4:545
-549.
Davidson A, Vermesh M, Lobo RA, Paulson RJ. Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model. Fertil Steril.1988; 49:516 -521.[Medline]
Fleetham JA, Pattinson HA, Mortimer D. The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization. Fertil Steril.1993; 59:192 -196.[Medline]
Fleming TP, Pratt HPM, Braude PR. The use of mouse preimplantation embryos for quality control of culture reagents in human in vitro fertilization programs: a cautionary note. Fertil Steril. 1987;47:858 -860.[Medline]
Parinaud J, Reme J-M, Monrozies X, Favrin S, Sarramon M-F, Pontonnier G. Mouse system quality control is necessary before the use of new material for in vitro fertilization and embryo transfer. J IVFET. 1987;4:56 -58.
Rinehart JS, Bavister BD, Gerrity M. Quality control in the in vitro fertilization laboratory: comparison of bioassay systems for water quality. J IVFET.1988; 5:335 -342.
Scott LF, Sundaram SG, Smith S. The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program. Fertil Steril.1993; 60:559 -568.[Medline]
World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm—Cervical Mucus Interaction. Cambridge: Cambridge University Press;1999 .
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