In order for labs to accurately monitor and assess their performance through the use of samples provided by an external source, such as an EQA scheme, the nature and quality of these samples must be as close to ‘real’ specimens as possible. Our aim at EQASRM is to provide realistic samples that are good quality, biologically safe and interesting to laboratory staff performing the assay.
We offer 11 schemes relevant to the field of reproductive medicine. Laboratories enrol in the schemes relevant to their unit. 4 samples for each scheme are sent quarterly, per financial year. Laboratories are then asked to submit their results within a set time frame. Results are to submitted online by the set closing date. Statistical and graphical analysis of all results are then made available for online viewing and download. Laboratories can see how their quantitative results and qualitative assessments compare to those of other labs in this field.
In conjunction with FSH, antral follicle count and ovarian volume, testing of Anti-Mullerian Hormone (AMH) levels has been shown to be a valuable marker in predicting a woman's ovarian reserve and function. An increasing number of IVF units are now requesting this test as part of the routine patient work up. As it is a relatively new assay, a need exists for inter-laboratory comparison in the form of participation in an EQA scheme as part of your laboratory's own in-house validation. We anticipate that evaluation of all the results will assist in the establishment of accurate reference ranges. Four screened serum samples are sent for testing. Specific information pertaining to each sample will be given to assist in clinical interpretation of results. In addition to submitting a numerical result, labs will also have the opportunity to add a short comment to each result. Statistical analysis will be performed on the results.
EQASRM creates three semen pools from carefully screened specimens, from which samples for testing are dispensed. We aim to provide samples with varying concentrations, all similar to those commonly assessed in the laboratory. The pools are fixed with a semen dilution fluid containing formaldehyde. The fourth sample sent for evaluation of concentration is a suspension of latex beads in serum.
The latex beads are of a similar diameter to that of a sperm head and are at a set concentration. By sending samples of this nature the laboratory is able to assess them as it would any sample it receives. Therefore, all variables such as mixing, pipetting, dilution, chamber differences and so on are tested.
These same semen samples are also used for morphological assessment by the lab. Laboratories create their own smears, stain and assess them according to their usual protocol. One pre-stained semen slide is also sent in an attempt to remove the ‘staining’ variable and compare labs merely on their criteria used for morphology assessment.
Samples of screened serum are sent to labs for detection of antisperm antibodies. The samples may be from a serum pool or from individual donors. Labs are asked to use their usual method of testing for antisperm antibodies and report on whether the level of antibodies detected was clinically significant. A positive and negative control are always sent in each distribution.
Sera from patients on gonadotrophin stimulated cycles is used for these samples. Samples are screened and pooled into 4 lots of specific concentration ranges. The ranges are reflective of those commonly seen in patients on stimulated cycles. Laboratories test these samples as per their biochemistry protocol.
Cytotoxicity testing is a crucial part of the IVF laboratory. EQASRM sends out a variety of samples in order for laboratories to test the sensitivity and validity of the assay they employ. The following are examples of samples that may be sent for testing:
- Embryo culture medium spiked with various toxins eg formaldehyde, chlorhexidine handwash, perfume etc.
- Embryo culture medium spiked with various products used in the IVF lab eg. propanediol, glycerol, lubricant, aqueous creams, handcleansers, etc.
- Products commonly used in the IVF lab: Cryopreservation straws, ET and IUI catheters, Semen Collection Devices, Condoms, mineral oil, etc.
The assessment of sperm vitality may be included as part of routine semen analysis but is especially important for samples with less than approximately 40% progressively motile sperm.
Our scheme is based on assessing the percentage of live spermatozoa by identifying those sperm with an intact cell membrane using dye exclusion. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in dead cells, allow entry of the stain.
For this scheme, 4 eosin-nigrosin stained semen smears are sent for analysis. Eosin is the stain that is taken up by cells with compromised membranes and the nigrosine increases the contrast between the background and the sperm heads. The slides should be examined with brightfield optics at x1000 magnification and oil immersion. Sperm with red or pink heads are considered dead whilst sperm with white heads are considered live. If the stain is limited to only a part of the neck region and the rest of the head is unstained, this is considered a “leaky neck membrane” and not a sign of cell death. These should be assessed as live. Labs are required to report the “percentage of live sperm” according to these criteria. The lower reference limit for vitality (live sperm) is 58%.
These slides can be retained for your staff training and IQC exercises.
This module provides an appropriate complement or alternative to the serum based "Indirect Anti-sperm Antibody Testing Scheme". Whilst the testing of serum samples for the presence of Anti-sperm Antibodies is still regarded as an important part of the assessment of an infertile couple, it's methodology differs from that used to test for sperm antibodies in semen. For this reason it is not feasible to send semen samples for determination of Anti-sperm antibody levels. This scheme provides footage of sperm/bead interactions in a similar fashion to our Sperm Motility Scheme, allowing users to view the footage and assess the percentage of motile sperm bound to beads. As with the other schemes, labs are asked to determine if the level of binding is "of clinical significance" based on the standards set by the WHO laboratory manual for the examination of human semen sperm-cervical mucus interactions 5th ed. Please note that binding restricted to the tail tip is not associated with impaired fertility and therefore users are asked to exclude this type of binding from their assessments.
It would not be possible to send ‘live’ semen samples to labs given factors such as delays in transportation and temperature maintenance. Therefore, EQASRM sends out footage of semen samples recorded during microscopic evaluation. Recordings of four samples are done at 37C°, using an inverted or phase contrast microscope. Six fields of view per sample are recorded, each lasting approximately 20-25 seconds. Labs are asked to report the motility of each sample using the WHO 5th edition, 3 category system. This is so results can be standardized and appropriately compared with others.
The assessment of sperm DNA fragmentation may be included as part of routine semen analysis but could be especially important for couples seeking fertility treatment. This scheme is designed for those laboratories assessing sperm DNA fragmentation using the Halosperm G2 assay, a method based upon the Sperm Chromatin Dispersion test. Each distribution contains video footage of slides prepared from 4 semen samples, with the footage showing the views seen using a brightfield microscope. Laboratories will be asked to determine proportion of sperm with DNA fragmentation according to their routine criteria, and whether the result was within the laboratory’s reference range. These DVD’s can be retained for your staff training and IQC exercises and results can also be entered into the EQASRM Internal QC module.
Footage of four Day2/3 and two blastocyst stage human embryos are recorded. The embryos are recorded whilst in culture medium microdroplets under oil at 37C°. The embryos are rotated and rolled using micropipettes as would be done in a routine assessment of embryo quality. Labs are asked to grade the embryo and, if appropriate, count the number of viable cells present. Other questions relating to each individual embryo are also asked in order to gain some insight into how labs assess their embryos and what their fate would be.
This scheme has been designed for users of time-lapse monitoring incubators. Time-lapse assessment of embryo morphokinetics is showing immense potential as an effective tool to identify embryos with higher implantation potential or as a de-selection tool for embryos displaying morphokinetic events associated with significantly lower rates of implantation such as direct cleavage (Rubio et al 2012) or abnormal syngamy (Wirka et al 2014). Embryo mophokinetic behaviour has also been shown to be associated with aneuploidy status (Campbell et al 2013).
Accurate determination and identification of cellular and developmental events is crucial for time-lapse imaging users. Currently there is limited data on within and between laboratory variability in this field. Participation in our "Embryo time-lapse" scheme would be of great benefit to users of this system for not only peer comparison but also for within lab monitoring using the IQC module.
Complete video footage from insemination to Day 5 or 6 of development for 2 embryos is sent on USB to participating labs. Video resolution is such that individual morphokinetic events can be identified easily using the pause/play function on Windows Media Player.
Three questions relating to specific timing of events requiring a numerical response are asked and statistics are performed on all results received. An additional three qualitative questions per embryo are also posed and these are summarised in tally form.
Click here to view the definition of morphokinetic events.
Sperm and Embryo footage is available on Windows VCD or DVD. Please note that the Windows VCD will only run on Windows Media Player Version 8 or above.