1 1Department of Obstetrics/Gynecology, Andrology/Embryology Laboratory, Oregon Health Sciences University, 2 Department of Physiology/Pharmacology, Oregon Health Sciences University, Portland, OR 97201 and 3 Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, OR 97006, USA
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Abstract |
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Key words: fecundity/frozen donor semen/sperm banking/sperm penetration assay/therapeutic insemination
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Introduction |
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The recruitment of donors for TI is an increasingly difficult process; at Oregon Health Sciences University (OHSU) only one of eight men initially evaluated is accepted as a donor (Mixon et al., 1989) similar to the 7087% drop-out or failed screening rates reported by others (Sidhu et al., 1997). The three principal reasons for this low acceptance rate are; lack of interest after the initial phone interview or after filling out the intake questionnaire; secondary to serology or family medical history; and on the basis of semen quality or post-thaw survival. Once admitted to a TI by donor programme, donors are often selected by recipients on the basis of physical characteristics, educational levels, hobbies and general interests as well as a history of reported pregnancy within a programme. Ideally, sperm banks should initially select donors on the basis of fertility potential rather than establishing fertility or non-fertility on the basis of reported pregnancies once in a programme (Thyer et al., 1999
). For this reason and because of the expense and effort involved in recruiting donors and maintaining a sperm bank, it is important to look for a reliable, prognostic test for selecting those donors that will give the best performance.
The sperm penetration assay (SPA) has been used for fertility potential measurement and male factor infertility evaluation for well over a decade (Overstreet et al., 1980; Binor et al., 1981
; Blazac and Overstreet, 1981
; Wolf and Sokoloski, 1982
; Martin and Taylor, 1983
; Wolf et al., 1983
; Irvine and Aitken, 1986
; Crister et al., 1987
; Kuzan et al., 1987
; Smith et al., 1987
; Corson, 1990
). Irvine and Aitken (Irvine and Aitken, 1986
) reported a significant difference in the SPA scores of individual frozen donor ejaculates that produced a cycle pregnancy compared to those that did not. Their fecundity correlation improved when sperm movement criteria were included. However, the accuracy and utility of the SPA as a prognostic indicator of donor fecundity in the context of TI by donor with frozen semen has not been established (Barratt et al., 1998
).
The biological basis for the SPA is dependent on two factors, the ability of the hamster egg to fuse with and subsequently decondense the head of human spermatozoa and the requirement that spermatozoa must undergo capacitation before fusing with the egg plasma membrane. Thus, the SPA provides a measure of the ability of the spermatozoon to complete physiological events that are required for normal fertilization (Yanagimachi et al., 1976; Aitken, 1994
).
The objective of the present work was to correlate SPA scores obtained from cryobanked semen with corresponding fecundities in a group of established sperm donors, thereby evaluating the efficacy of the SPA in screening potential donors for sperm banking. It should be emphasized that the SPA was performed after the initial donor screening on the frozen aged semen and not on the fresh specimen. Hopefully, this approach would account for any hidden cryo damage that might have occurred during the freezethaw process. This investigation was made possible by the optimized SPA quality control technology that routinely measures sperm penetrating ability from frozenthawed semen. Since the processing conditions of the optimized SPA were originally developed to distinguish the infertile from fertile population rather than the relative performance among just the fertile population, conditions were also altered here in an effort to provide enhanced assay sensitivity.
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Materials and methods |
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Screening protocol
Eleven donors underwent a standardized screening protocol, including physical examination, and review of medical, family and infectious disease history. All donors met minimum semen criteria defined as >70% normal morphology (as defined by the World Health Organization, 1992), sperm count >60x106/ml, post-thaw motility (demonstrating some forward progression) >25% and at least 50% cryosurvival. The storage age for donor samples tested ranged from 1.1 to 5.1 years (mean 3.4 years). The majority of IUI patients underwent initial evaluations at OHSU. They were carefully screened to eliminate any detectable pathology. Patients with risk factors for tubal disease were screened with hysterosalpingography and were referred for treatment or to in-vitro fertilization (IVF) if there was evidence of tubal occlusion. Ovulatory dysfunction was treated with either clomiphene citrate or gonadotrophin therapy. This study includes all IUIs performed from October 1990 to February 1998. All inseminations were performed at OHSU on the day after a luteinizing hormone (LH) surge was detected by a urine predictor kit. Pregnancy was defined as a positive pregnancy [urine or serum human chorionic gonadotrophin (HCG)] test or a verbal report from the patient if she was followed by an outside provider.
Statistical analysis
Data sets were analysed using statistical software from SAS Institute Inc. (Gary, NC, USA). A Pearson correlation coefficient or a linear regression probability <0.05 was considered to be significant. SPA run values were included in this study only if the two control values included in assay passed established Westgard rules of compliance (Westgard et al., 1981). Westgard defined seven rules for accepting or rejecting assay outcomes based on a comparison of control values with the standard deviation of the prior corresponding controls plotted on a LeveyJennings chart. After every SPA test run all the previously obtained values for a control series are used to recalculate a new or `running' standard deviation. By adding the previous run control value, the increased number of values improves the definition of acceptable limits.
Sperm cryopreservation
Semen samples, collected by masturbation, were liquefied for approximately 30 min at 37°C before a semen analysis was conducted. Samples were frozen by one of two methods.
Conventional method (n = 861)
One volume of semen was diluted with 3 volumes of cryoprotectant, 211 mmol/l TES (N-tris [hydroxymethyl] methyl-2-amino-ethane sulphonic acid), 96 mmol/l Tris (hydroxymethyl-aminomethane), 11 mmol/l dextrose, and 1% penicillinstreptomycin solution to which was added 20% fresh hen yolk, TEST Yolk Buffer (TYB) (Irvine Scientific, Santa Ana, CA, USA) modified by the addition of glycerol to a concentration of 29.6%. The final glycerol concentration in the semen/cryoprotectant mixture was 7.4%.
IUI-Ready method (n = 44)
Semen was centrifuged at 350 g for 20 min through a 507090% Percoll (Pharmacia, Uppsala, Sweden) gradient. The pellet was washed once and resuspended in IUI-Ready Cryoprotectant [Tyrode's solution with albumin and pyruvate (TALP), HEPES6% glycerol2% sucrose] to approximate a motile sperm concentration of 40x106 per ml. In both methods, diluted samples were transferred to pre-labelled Nunc (Nalge Nunc International, Naperville, IL, USA) cryotubes (including a test vial to be thawed for cryosurvival), placed on wands submerged in a beaker of water at ambient temperature and held at 4°C for 1 h. Samples were then exposed to liquid nitrogen vapours for 30 min and immersed in liquid nitrogen for 5 min prior to placement in permanent storage locations. A minimum of 24 h after freezing, a test vial was thawed, the sperm motility determined, and the number of motile spermatozoa per freeze vial was calculated. This laboratory found in a recent study that fecundity rates using conventional compared to IUI ready methods were statistically identical (Mixon et al., 1998
).
Sperm thaw and preparation for IUI
One hour before the scheduled insemination, vials were removed from liquid nitrogen and placed at room temperature for 5 min, then at 37°C for 10 min. Conventionally frozen samples were washed by one of two methods.
Wash/resuspension method
The thawed semen was mixed with two volumes of wash medium [human tubal fluid (HTF)-HEPES + 1% human serum albumin (HSA)] in a 15 ml conical centrifuge tube, centrifuged for 7 min at 350 g before the resulting pellet was resuspended in 2 volumes of wash medium and centrifuged in the same manner. The final pellet was resuspended in 0.4 ml of wash medium.
Gradient centrifugation method
The thawed semen was layered on top of a 507095% Percoll (Pharmacia) gradient and centrifuged at 350 g for 25 min. The resulting pellet was resuspended in 3.0 ml of wash medium, centrifuged at 350 g for 7 min. The final pellet was resuspended in 0.4 ml of wash medium. For both methods, the total number of motile spermatozoa in the final preparation was calculated.
Sperm thaw and preparation for SPA
A single vial from three different ejaculates from each donor were thawed in a 4°C waterbath. All vials tested in the SPA were frozen by the conventional method (above). An aliquot was immediately analysed to determine post-thaw motility and the rest of the sample was held for an additional 18 h at 4°C in preparation for the SPA. The next morning the 4°C semen samples were thermal shocked by the rapid addition of 6 ml 37°C Biggers, Whitten, and Whittingham's buffer containing 0.3% human serum albumin (BWW-0.3% HSA) (Biggers et al., 1971). Samples were centrifuged at 300 g for 10 min, aspirated and resuspended in 3.0 ml of BWW-0.3% HSA before distribution into six 12x75 mm culture tubes, and centrifuged at 300 g for 10 min. Swim-up spermatozoa were harvested after a 90 min incubation at 37°C, centrifuged again at 300 g for 10 min, and resuspended to normalize the final concentration to 5.0 (or 1.0, 0.5, or 0.25 when indicated)x106 motile spermatozoa/ml.
Collection, processing and storage of semen for quality control
Semen samples that contained over 200x106 motile spermatozoa were collected from individuals with established SPA levels and diluted with an equal volume of TYB containing 8% glycerol (Johnson et al., 1995). The sample was aliquotted into 0.5 ml French straws and sealed with putty. Straws were placed horizontally in a Petri dish, incubated at 4°C and 20°C for 10 min each, frozen in liquid nitrogen vapour for 30 min, and finally placed in liquid nitrogen for long-term storage. Before each SPA run, straws were retrieved from storage, immersed in a 4°C water bath for 18 h and then rapidly transferred to a 37°C water bath for 3 min. The sample was dispensed from the straw into a conical tube containing 6 ml of 37°C buffer (BWW-0.3% HSA) and processed as described above.
Hamster ova preparation
Golden Syrian hamsters received ovarian stimulation after which the oviducts were excised and transferred to the laboratory in 04°C BWW-0.3% HSA (Johnson et al., 1990). Cumulus masses were retrieved and placed in medium with 1 mg/ml hyaluronidase. The zonae pellucidae were then enzymatically digested in medium with 1 mg/ml trypsin. Following multiple washes, 20 zona-free ova were added to twin 50 µl drops containing motile spermatozoa, covered with silicone oil and incubated at 37°C for 3.5 h in 95% humidified air. After incubation, ova were washed to remove loosely bound spermatozoa, carefully placed on a glass slide, and topped with a coverslip. Phase-contrast microscopy at x400 was used to confirm the presence of decondensed sperm heads associated with tails, interpreted as a positive penetration. Most patient specimens tested by the optimized SPA will have had all ova penetrated by multiple spermatozoa. Donor scores were expected to average 35 penetrated spermatozoa in every ovum incubated (Johnson et al., 1990
). Consequently, SPA scores were expressed as the mean number of penetrations/ovum (p/o, total penetrations/total number ova incubated, previously described as sperm capacitation index or SCI).
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Results |
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Discussion |
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The testing of fertility potential of frozen semen has been reported previously (Irvine and Aitken, 1986; Crister et al., 1987
) as has the use of frozen semen for quality control purposes (Smith et al., 1987
; Johnson et al., 1995
) where samples are run multiple times, thereby defining both inter-assay and intra-ejaculate variability. In the present study, these coefficients of variation for QC samples ranged from 9 to 31% and were consistent with prior reports (Johnson et al., 1990
, 1995
). Here we also established that SPA scores on three ejaculates from each of 11 donors showed a remarkable degree of consistency. The inter-ejaculate variability ranged from 11 to 67%, similar to the variability previously reported for fresh semen from four donors over time (2142%) (Johnson et al., 1990
).
The prognostic significance of the SPA in the evaluation of male fertility potential has been challenged. A number of investigators have reported on the positive correlation with male infertility (in vitro and in vivo) (Wolf et al., 1983; Smith et al., 1987
; Johnson et al., 1990
, 1991
); however, others have been critical, indicating that the SPA is a poor indicator of sperm function (Corson, 1990
) or has an unacceptably high percentage of false negative results (Kuzan et al., 1987
; Corson, 1990
). SPA studies performed on small numbers of patients may contribute to the conflicting results. Also, since laboratories do not follow a standardized protocol, or employ standardized quality control procedures such as the use of internal controls as described herein, it is virtually impossible to compare results between groups. The present study supports the thesis that the SPA provides a reliable and reproducible approach to the analysis of sperm function in vitro (Overstreet et al., 1980
; Wolf et al., 1983
; Smith et al., 1987
; Johnson et al., 1990
; Aitken, 1994
).
Penetration scores generated by the improved SPA, as employed here, have been assigned clinically significant predictive values based on the testing of pregnancy proven donors (Johnson et al., 1990). A threshold value for the SPA was established at >5.0 p/o (derived from a value 2 SD below the fertile population mean at 35.0 p/o ± 15 SD, n = 30). In the present group of 11 pregnancy proven donors only two showed mean SPA scores near the threshold values defined above. Donor 1 had the lowest fecundity with a mean SPA score of 8.7 p/o (range 2.714.3) while donor 3 with the second lowest fecundity had a mean SPA score of 11.0 p/o (range 9.413.1). The remaining nine donors showed mean SPA scores between 23 and 66.6 p/o. This finding is consistent with the expectations of proven donors as measured on non-frozen semen (Johnson et al., 1990
, 1995
), but contradictory results have been reported by others (Binor et al., 1981
; Blazac and Overstreet, 1981
) using frozen donor semen for predicting fertility status.
The correlation observed in this study between increasing fecundity and mean SPA scores was obtained at several sperm concentrations ranging from a low at 0.25 to a high at 5.0x106/ml, the standard concentration. The non-linear relationships between fecundity and mean SPA scores suggest that it may be efficacious to evaluate such a cohort of men with relatively high penetrating spermatozoa at lower inseminating sperm concentrations. At standard concentrations the number of penetrations per ovum may plateau secondary to the saturation of sperm binding sites on the oocyte plasma membrane or to saturation of the egg cytoplasm. Differences in the kinetics of capacitation may also be involved, which are known to vary among proven fertile donors (Wolf and Sokoloski, 1982; Martin and Taylor, 1983
; Smith et al., 1987
). By reducing the sperm concentration, penetration rates became more highly correlated to donor fecundity. Based on these findings, a reasonable SPA threshold value to distinguish potentially good from poor donors at a sperm concentration of 0.25x106 motile spermatozoa/ml can be proposed at 2 p/o. All four donors with a fecundity <0.06 had SPA scores <2 p/o and all seven donors with a fecundity >0.06 had SPA scores >2 p/o. It is interesting to note that the optimized SPA and the modified Micro-SPA (Johnson et al., 1991
) as well as this `frozen donor' SPA system have evolved to utilize different sperm concentrations. Thus, customization is required to position the assay discrimination value at the most sensitive portion of the penetration curve, to best separate men with different fertility potential.
The therapeutic donor insemination programme has provided a unique opportunity to investigate the predictive value of in-vitro tests of sperm function. We conclude that a standardized SPA protocol on cryopreserved spermatozoa can serve as a valuable supplement to the more traditional tests of semen quality in efforts designed to evaluate donor fecundity. The selection of high fecundity donors is a significant issue in maximizing TI by donor pregnancy rates. Currently there are no guidelines or minimum performance standards for donors. Some retrospective studies suggest dropping donors who had not produced a pregnancy by 12 cycles (McGowan et al., 1983). Thyer and co-workers (Thyer et al., 1999
) analysed the first 40 cycles in 20 TI donors. The fecundity was constant with normal distribution in 20 cycles, but after only 15 cycles, 80% did not deviate by more than one quartile; donor performance at 15 cycles was predictive of performance at 40 cycles. They concluded that the individual analysis of donor fecundity every 15 cycles is important for sperm banks to consider and would justify dropping donors whose performances are poor.
A substantial cost benefit can be realized by both the cryobank and semen recipients if the SPA is used routinely to eliminate substandard donors. The cryobank would spend an additional fee for SPA testing; however, when performed as an initial donor screen such testing can preclude the costs and professional time invested in qualifying family medical history, physical examination and testing for infectious diseases. Moreover, as this technology evolves it may become possible to freeze fewer spermatozoa per vial from high SPA donor specimens. Diluting inseminations on the basis of sperm quality rather than quantity would mean fewer specimens would need to be collected and processed by the cryobank.
We can use the 11 donors in the present study and their 905 TI cycles as a model to predict the potential cost benefit for patients. The four poorest donors (fecundity 0.05 and SPA <2.0 p/o) collectively achieved 11 pregnancies in 204 cycles. If they were never used the remaining seven donors would achieve the same number of pregnancies in just 92 cycles (0.12 fecundity and SPA >2.0 p/o). Considering the demand in the USA for donor insemination has been estimated to be >270 000 units per year (Crister, 1998), the overall cost benefit could be substantial.
In summary we have demonstrated a positive relationship between sperm function measurements on cryopreserved donor semen and corresponding donor fecundities. By modifying SPA conditions to use a lower concentration of motile spermatozoa, the assay became more predictive of fertility potential. Since additional results are required before pre-screening donors becomes clinically relevant, we have initiated a prospective controlled study testing a broader spectrum of donors in our TI programme.
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Acknowledgments |
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Notes |
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5 To whom correspondence should be addressed at: Andrology/Embryology Laboratory, Oregon Health Sciences University,1750 S.W. Harbor Way, Suite 100, Portland, OR 97201-5164, USA
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References |
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Submitted on May 24, 1999; accepted on October 18, 1999.