Optimized Sperm Penetration Assay 

The photo on the left contains a  zona-free hamster egg with more than 20 penetrated sperm, with swollen heads. To see a larger display of this picture, click here. Please note that the larger picture is 388K; it may take some time to download.

The population suitable for testing includes:

• Infertile couples experiencing unexplained infertility
• Normozoospermic patients that have failed >3 IUI attempts
• Male factor patients considering IVF

There are dramatic differences in the Optimized SPA compared to other SPA protocols. We have established unique controlled conditions for processing both the sperm and ova used in this test. Cold temperature storage of sperm (4°C for 42 hours) effectively synchronizes capacitation resulting in very high sperm penetration rates. Since most patientsí specimens penetrate all the ova we score the average number of penetrations per ovum rather than the percentage of ova penetrated.  Other SPA systems typically have 0, 1, or 2 sperm penetrating each egg compared to the 10, 30, or 50 penetrations/egg achieved in the Optimized SPA, (see photo above).

Local Lab semen collection Vs. Mail-in SPA results are identical.  Our routine ìin houseî SPA protocol refrigerates all semen for 2 days.  We receive overnight Fed Ex refrigerated semen from outside laboratories and place them in our refrigerator (with in house specimens) to complete the 2nd day of storage.  We find no differences when we ship split specimens back to our laboratory.

Signal to noise ratio is an important consideration in any diagnostic test including the SPA. As in IVF, when good quality eggs are not all fertilized when exposed to the same sperm population, hamster eggs also show different degrees of penetrability to human sperm. Therefore, any system using hamster eggs to judge sperm penetration rates must overcome the differences (noise) inherent in hamster egg quality. Since we score the average number of penetrations per ovum rather than the percentage of ova penetrated, we largely overcome this source of noise. Also, the high penetration rates of the optimized SPA allow better statistical differentiation between the high scores of the pregnancy proven donors and low scores of the infertility patients. Thus, the "gray zone" between fertile and infertile values that makes interpretation difficult is minimized. 

fig caption: This graph depicts low, medium, and high results from 3 different patient semen specimens. The resulting penetrations in each of ten ova are graphed (more are used in actual testing) to show how we derive the average penetrations per ovum. The small box in the lower left represents the limited range of values achieved testing the same semen using routine SPA technology. Even when all ova (100%) are penetrated more clinical information is available when scores are reported as average penetrations per ovum. This increased assay sensitivity significantly lowers the occurrence of false negative results (poor SPA yet good fertilization, associated with previous SPA protocols) without contributing to false positives. 

Patients achieving the following SPA scores have an increased statistical likelihood of being included in the described groups. 

>10 (Directly comparable to scores from the fertile population, 35.0 mean ± 15.0 standard deviation, range 68.0 to 11.0) Expect normal fertilization in routine IVF and IUI when suitable total motile sperm are recovered. 

> 10 and > 5 ("Subfertile", below all donor scores) Expect normal in vitro fertilization rates when the insemination concentrations are increased; the closer the SPA score approaches 5 consider ICSI to optimize IVF; after 3 cycles of unsuccessful IUI patients should consider IVF (especially when the inseminations were of good quality).

< 5 ("Abnormal", 2 standard deviations below scores from the fertile population) Expect decreased in vitro fertilization rates or no fertilization with routine IVF. Some patientís sperm will not fertilize even when increased insemination concentrations are used. ICSI is strongly recommended. IUI patients should consider ICSI.

Assumptions:

• the semen quality (especially % motility) presented at the time of IVF or IUI is similar to the semen quality submitted for the SPA
• both partners are free from sperm antibodies
• female factors are not suspected for IUI couples
• good quality eggs are retrieved for IVF

The Micro-SPA is a specialized SPA that was developed for those patients who experience low counts or poor motility. By normalizing test conditions to utilize only one tenth the number of sperm needed in the Optimized SPA, test results can be directly compared from patients that could not otherwise be tested. Instead of incubating sperm and ova in a culture dish, this assay maintains high penetration rates by concentrating a low number of sperm about the ova in a microfuge tube. Since sperm from patients with poor quality semen have a fair chance, this test more accurately predicts outcome on the basis of sperm function rather than sperm quantity. 

Reports of the Optimized and the Micro-SPA use a specific value set and scale to compare results. The graphs reflect the statistical probability that the patients' results will be included in the fertile range. The Optimized SPA will also show the relative probability of fertilizing at least a third of the human ova in a routine IVF cycle. 

All of these techniques have now been standardized to provide a more statistically precise and reproducible analytical assessment of sperm function. These are just some of the reasons why the Optimized SPA is now regarded as one of the best diagnostic tests of male fertilizing potential. 

Innovative Advantages of the Optimized SPA 

1. Low temperature semen conditioning in TEST-Yolk Buffer (TYB) dramatically increases assay sensitivity.^(1-3)
2. Aliquots of frozen semen are employed in a unique approach to Quality Control evaluation.⁽⁴⁾
3. A Micro-SPA is available specifically for low recovery, oligozoospermic patients.⁽⁵⁾
4. Frozen semen can be tested.^(6,9)
5. Only fresh ova are used (not frozen).
6. Cold ova handling preserves ova sensitivity.⁽⁷⁾
7. Optimized TYB formulation enhances the capacitation process.⁽⁸⁾
8. Overnight mailing at low temperature is consistent with the optimized technology and supports semen collection off campus.⁽¹⁾

1. Johnson A.R., et al. (1990) In CRC Handbook of the Laboratory Diagnosis and Treatment of Infertility. (Eds. Keel and Webster) Boca Raton, CRC Press, p. 135
2. Jacobs, B.R., et al. (1995) Fertility & Sterility 63:1064

 
3. Soffer Y., et al. (1992) Fertility & Sterility 58:556

 
4. Johnson A.R., et al. (1995) Fertility & Sterility 64:832

 
5. Johnson A.R., et al. (1991) Fertility & Sterility 56:528

 
6. Johnson A.R. and Hessel P., (1995) Am. Soc. Rep. Med., Suppl. Abst# P-168, p. 171

 
7. Syms A.J., et al. (1985) Fertility & Sterility 43:766

 
8. Weathersbee P. and Johnson A.R., (1995) Fertility & Sterility 63:925
9. Navarrete T.,  Johnson A.R., et al. (2000) Human Reproduction vol 15: no2  p. 344