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Motility longevity of sperm samples processed for intrauterine insemination*
Vol. 58, No.2, August 1992
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright rj) 1992 The American Fertility Society
Motility longevity of sperm samples processed for intrauterine insemination*
Johan Denil, M.D. t Dana A. Ohl, M.D.t+ William W. Hurd, M.D.§
Alan C. Menge, Ph.D.§11 Melissa R. Hiner, M.L.T. (ASCP)§II
University of Michigan Medical Center, Ann Arbor, Michigan
Sperm motility plays an extremely important role in success from intrauterine insemination (lUI). The ability to maintain motility over time, termed motility longevity, may also be an important factor because insemination and ovulation may not coincide exactly. Because sperm obtained by electroejaculation exhibits many functional abnormalities, including poor viability and survival (1), poor motility longevity may contribute to the low pregnancy rates (PRs) seen with electroejaculation/IUI. Male factor infertility, diagnosed on the basis of abnormal semen analyses, may also be associated with decreased longevity. To determine if sperm motility longevity is decreased in men with abnormal semen analyses and in men undergoing electroejaculation, sperm motility was evaluated before and after processing for lUI and again 24 hours later. MATERIALS AND METHODS
Sperm samples from 74 men were evaluated. Normal semen samples by World Health Organization (WHO) criteria (2) were obtained from 19 men (normal group). Most of these men were from couReceived October 15, 1991; revised and accepted April 7, 1992.
* Presented at the 86th Annual Meeting of the American U rological Association, Toronto, Ontario, Canada, June 2 to 6, 1991. t Section of Urology, Department of Surgery. :j: Reprint requests: Dana A. Ohl, M.D., University of Michigan Medical Center, 1500 E. Medical Center Drive, Box 0330, Section of Urology, 2916 Taubman Center, Ann Arbor, Michigan 481090330. § Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology. II Laboratory for Assisted Reproductive Technology.
436
Denil et al.
Communications-in-brief
pIes being treated with lUI for unexplained infertility (n = 6) or isolated female factor infertility (n = 6), and the others were healthy volunteers (n = 7). The decision to place patients in the normal group was based on WHO criteria of the semen analysis and lack of an identifiable male factor. Thirteen abnormal semen samples were obtained from infertile couples being treated with lUI for identified male factor infertility (male factor group). Forty-two samples were obtained by electroejaculation as described previously (3) from men with anejaculation (electroejaculation group) because of spinal cord injury (n = 29), retroperitoneal lymphadenectomy (n = 10), diabetic neuropathy (n = 2), and idiopathic anejaculation (n = 1). After semen analysis was performed, motile sperm were isolated using a swim-up technique. The retrograde semen fractions from electroejaculation were first separated from the urine by centrifugation at 400 X g for 8 minutes and the supernatant discarded. The pellet was resuspended in 0.5 mL of Ham's FlO solution (GIBCO, Grand Island, NY) with 20 mmol Hepes buffer (Sigma Chemical Company, St. Louis, MO) and 5% bovine serum albumin (BSA). Aliquots of the retrograde pellets and antegrade semen were placed below 4.5 mL of Ham's F-10 solution with 20 mmoljHepes buffer and 1% BSA and incubated in test tubes slanted 45 ° from vertical for 60 minutes at 37°C. The supernatant was aspirated and centrifuged twice for 8 minutes at 400 X g and resuspended in the medium. The processed antegrade and retrograde fractions obtained by electroejaculation were pooled. If one fraction or another was of very low quality, it was discarded.
Fertility and Sterility
100 80
j
60 40 20
A.Motility
--
~.-,"~ ...•... " , t .....' ". . . . t
Normal MaleFactor Blectroejaculation
the electroejaculation group was less than that of both the normal group and the male factor group (P < 0.05, Fig. 2).
.1..* '1t •........ ········...t
DISCUSSION
0
B. Forward Progress
100 80
I
Q..
60 40
...... .', t ~ ···\tt
20
C. Forward Speed
*
e
~t ..... . ....~~'. t ·It
en
Processing Before
After
24 b After
Figure 1 Sperm motility characteristics of samples from normal (n = 19), male factor (n = 13), and electroejaculation (n = 42) samples before, immediately after, and 24 hours after (24 h after) processing. Values are means ± SE. ('" versus before, t versus after, P < 0.05, 2-factor repeated measures ANOVA.)
After the swim-up, samples were analyzed by microscopic analysis by a laboratory technician for count, percent motility, percent forward progression, and forward speed (scale: 1 to 4). An aliquot of each sample was diluted with media to a final concentration of 250,000 sperm/mL. After incubation at 37°C for 24 hours, the sperm motility variables were reanalyzed. The motility longevities at 24 hours were expressed as the percent of postprocessing motility and compared using a two-factor repeated measure ANOVA, followed by either a Scheffe's F test or a Neuman-Keuls multiple range test to determine individual differences. Probability values of <0.05 were considered significant.
Intrauterine insemination has become increasing more common in the treatment of male factor infertility. The present study shows that sperm obtained for lUI from men with abnormal semen analyses and from men undergoing electroejaculation have reduced motility longevity when compared with normal sperm. This suggests that accurate timing of insemination to ovulation (24 to 36 hours after urinary detection of the luteinizing hormone [LH] surge) is very important in these patients. In support of this is the observation that lUI for the treatment of oligoasthenozoospermia performed 12 to 24 hours after urinary detection of the LH surge rarely results in pregnancy (4), whereas lUI performed 24 to 36 hours after the LH surge results in a much higher PR (22%) (5). Although repeated inseminations on 2 consecutive days may compensate for less exact timing of artificial insemination, both monetary and time expenditure is also increased. This increased cost may be substantial when electroejaculation is required. Another consideration is timing of specimen collection. Specimen collection for lUI is simple for most patients, but in the case of electroejaculation, a physician with technical support and occasionally anesthetic support is necessary. Because motility
80 60
.£ =i03
::E
40
-g
RESULTS
The separation procedure effectively isolated the motile sperm. The percent motility, percent forward progression, and the forward speed were significantly improved in all groups. After 24 hours, all three motility variables were significantly decreased in all groups (Fig. 1). However, the 24-hour motility longevity in the male factor group was less than that of the normal group, and the motility longevity of
Vol. 58, No.2, August 1992
.§
20
Q)
~
~
0 Normal
Male Factor
Electroejaculation
Figure 2 Motility longevity of sperm 24 hours after processing expressed as a percentage of the motility immediately after processing for lUI. Values are means ± SE. Differences significant between the groups: * versus normal group, t versus male factor group, P < 0.05, one-factor ANOVA.
Deni! et at.
Communications-in-brief
437
longevity of sperm obtained by electroejaculation is reduced even more than sperm obtained by masturbation, the specimen should be obtained as near to the time of ovulation as possible, and the time between obtaining the specimen and insemination should be minimized.
detection of the LH surge or as close to the time of ovulation as possible. Key Words: Sperm motility, sperm survival, infertility, intrauterine insemination, electroejaculation, ovulation timing. REFERENCES
SUMMARY
This study demonstrates that sperm from men with male factor infertility and sperm obtained by electroejaculation have reduced motility longevity when compared with normal specimens. After 24 hours, normal samples lost only 34 % of initial motility, whereas male factor patients lost 48%, and electroejaculation patients dropped 66%. Based on these data and previous clinical studies of insemination timing, it is recommended that sperm retrieval and artificial insemination for male factor infertility, especially when electroejaculation is necessary, be performed 24 to 36 hours after urinary
438
Denil et al.
Communications-in-brief
1. Denil J, Ohl DA, Menge AC, Keller LM, McCabe M. Functional characteristics of sperm obtained by electroejaculation in men with anejaculatory infertility. J UroI1992;147:69-72. 2. World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge: The Press Syndicate ofthe University of Cambridge, 1987; Appendix 1A:27. 3. Ohl DA, Denil J, Bennett CJ, Randolph JF, Menge AC, McCabe M. Electroejaculation following retroperitoneal lymphadenectomy. J UroI1991;145:980-3. 4. Ho P, Poon IML, Chan SYW, Wang C. Intrauterine insemination is not useful in oligoasthenozoospermia. Fertil Steril 1989;51:682-4. 5. Kerin JF, Kirby C, Peek J, Jeffrey R, Warnes GM, Matthews CD, et al. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet 1984;1:533-5.
Fertility and Sterility
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright rj) 1992 The American Fertility Society
Motility longevity of sperm samples processed for intrauterine insemination*
Johan Denil, M.D. t Dana A. Ohl, M.D.t+ William W. Hurd, M.D.§
Alan C. Menge, Ph.D.§11 Melissa R. Hiner, M.L.T. (ASCP)§II
University of Michigan Medical Center, Ann Arbor, Michigan
Sperm motility plays an extremely important role in success from intrauterine insemination (lUI). The ability to maintain motility over time, termed motility longevity, may also be an important factor because insemination and ovulation may not coincide exactly. Because sperm obtained by electroejaculation exhibits many functional abnormalities, including poor viability and survival (1), poor motility longevity may contribute to the low pregnancy rates (PRs) seen with electroejaculation/IUI. Male factor infertility, diagnosed on the basis of abnormal semen analyses, may also be associated with decreased longevity. To determine if sperm motility longevity is decreased in men with abnormal semen analyses and in men undergoing electroejaculation, sperm motility was evaluated before and after processing for lUI and again 24 hours later. MATERIALS AND METHODS
Sperm samples from 74 men were evaluated. Normal semen samples by World Health Organization (WHO) criteria (2) were obtained from 19 men (normal group). Most of these men were from couReceived October 15, 1991; revised and accepted April 7, 1992.
* Presented at the 86th Annual Meeting of the American U rological Association, Toronto, Ontario, Canada, June 2 to 6, 1991. t Section of Urology, Department of Surgery. :j: Reprint requests: Dana A. Ohl, M.D., University of Michigan Medical Center, 1500 E. Medical Center Drive, Box 0330, Section of Urology, 2916 Taubman Center, Ann Arbor, Michigan 481090330. § Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology. II Laboratory for Assisted Reproductive Technology.
436
Denil et al.
Communications-in-brief
pIes being treated with lUI for unexplained infertility (n = 6) or isolated female factor infertility (n = 6), and the others were healthy volunteers (n = 7). The decision to place patients in the normal group was based on WHO criteria of the semen analysis and lack of an identifiable male factor. Thirteen abnormal semen samples were obtained from infertile couples being treated with lUI for identified male factor infertility (male factor group). Forty-two samples were obtained by electroejaculation as described previously (3) from men with anejaculation (electroejaculation group) because of spinal cord injury (n = 29), retroperitoneal lymphadenectomy (n = 10), diabetic neuropathy (n = 2), and idiopathic anejaculation (n = 1). After semen analysis was performed, motile sperm were isolated using a swim-up technique. The retrograde semen fractions from electroejaculation were first separated from the urine by centrifugation at 400 X g for 8 minutes and the supernatant discarded. The pellet was resuspended in 0.5 mL of Ham's FlO solution (GIBCO, Grand Island, NY) with 20 mmol Hepes buffer (Sigma Chemical Company, St. Louis, MO) and 5% bovine serum albumin (BSA). Aliquots of the retrograde pellets and antegrade semen were placed below 4.5 mL of Ham's F-10 solution with 20 mmoljHepes buffer and 1% BSA and incubated in test tubes slanted 45 ° from vertical for 60 minutes at 37°C. The supernatant was aspirated and centrifuged twice for 8 minutes at 400 X g and resuspended in the medium. The processed antegrade and retrograde fractions obtained by electroejaculation were pooled. If one fraction or another was of very low quality, it was discarded.
Fertility and Sterility
100 80
j
60 40 20
A.Motility
--
~.-,"~ ...•... " , t .....' ". . . . t
Normal MaleFactor Blectroejaculation
the electroejaculation group was less than that of both the normal group and the male factor group (P < 0.05, Fig. 2).
.1..* '1t •........ ········...t
DISCUSSION
0
B. Forward Progress
100 80
I
Q..
60 40
...... .', t ~ ···\tt
20
C. Forward Speed
*
e
~t ..... . ....~~'. t ·It
en
Processing Before
After
24 b After
Figure 1 Sperm motility characteristics of samples from normal (n = 19), male factor (n = 13), and electroejaculation (n = 42) samples before, immediately after, and 24 hours after (24 h after) processing. Values are means ± SE. ('" versus before, t versus after, P < 0.05, 2-factor repeated measures ANOVA.)
After the swim-up, samples were analyzed by microscopic analysis by a laboratory technician for count, percent motility, percent forward progression, and forward speed (scale: 1 to 4). An aliquot of each sample was diluted with media to a final concentration of 250,000 sperm/mL. After incubation at 37°C for 24 hours, the sperm motility variables were reanalyzed. The motility longevities at 24 hours were expressed as the percent of postprocessing motility and compared using a two-factor repeated measure ANOVA, followed by either a Scheffe's F test or a Neuman-Keuls multiple range test to determine individual differences. Probability values of <0.05 were considered significant.
Intrauterine insemination has become increasing more common in the treatment of male factor infertility. The present study shows that sperm obtained for lUI from men with abnormal semen analyses and from men undergoing electroejaculation have reduced motility longevity when compared with normal sperm. This suggests that accurate timing of insemination to ovulation (24 to 36 hours after urinary detection of the luteinizing hormone [LH] surge) is very important in these patients. In support of this is the observation that lUI for the treatment of oligoasthenozoospermia performed 12 to 24 hours after urinary detection of the LH surge rarely results in pregnancy (4), whereas lUI performed 24 to 36 hours after the LH surge results in a much higher PR (22%) (5). Although repeated inseminations on 2 consecutive days may compensate for less exact timing of artificial insemination, both monetary and time expenditure is also increased. This increased cost may be substantial when electroejaculation is required. Another consideration is timing of specimen collection. Specimen collection for lUI is simple for most patients, but in the case of electroejaculation, a physician with technical support and occasionally anesthetic support is necessary. Because motility
80 60
.£ =i03
::E
40
-g
RESULTS
The separation procedure effectively isolated the motile sperm. The percent motility, percent forward progression, and the forward speed were significantly improved in all groups. After 24 hours, all three motility variables were significantly decreased in all groups (Fig. 1). However, the 24-hour motility longevity in the male factor group was less than that of the normal group, and the motility longevity of
Vol. 58, No.2, August 1992
.§
20
Q)
~
~
0 Normal
Male Factor
Electroejaculation
Figure 2 Motility longevity of sperm 24 hours after processing expressed as a percentage of the motility immediately after processing for lUI. Values are means ± SE. Differences significant between the groups: * versus normal group, t versus male factor group, P < 0.05, one-factor ANOVA.
Deni! et at.
Communications-in-brief
437
longevity of sperm obtained by electroejaculation is reduced even more than sperm obtained by masturbation, the specimen should be obtained as near to the time of ovulation as possible, and the time between obtaining the specimen and insemination should be minimized.
detection of the LH surge or as close to the time of ovulation as possible. Key Words: Sperm motility, sperm survival, infertility, intrauterine insemination, electroejaculation, ovulation timing. REFERENCES
SUMMARY
This study demonstrates that sperm from men with male factor infertility and sperm obtained by electroejaculation have reduced motility longevity when compared with normal specimens. After 24 hours, normal samples lost only 34 % of initial motility, whereas male factor patients lost 48%, and electroejaculation patients dropped 66%. Based on these data and previous clinical studies of insemination timing, it is recommended that sperm retrieval and artificial insemination for male factor infertility, especially when electroejaculation is necessary, be performed 24 to 36 hours after urinary
438
Denil et al.
Communications-in-brief
1. Denil J, Ohl DA, Menge AC, Keller LM, McCabe M. Functional characteristics of sperm obtained by electroejaculation in men with anejaculatory infertility. J UroI1992;147:69-72. 2. World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge: The Press Syndicate ofthe University of Cambridge, 1987; Appendix 1A:27. 3. Ohl DA, Denil J, Bennett CJ, Randolph JF, Menge AC, McCabe M. Electroejaculation following retroperitoneal lymphadenectomy. J UroI1991;145:980-3. 4. Ho P, Poon IML, Chan SYW, Wang C. Intrauterine insemination is not useful in oligoasthenozoospermia. Fertil Steril 1989;51:682-4. 5. Kerin JF, Kirby C, Peek J, Jeffrey R, Warnes GM, Matthews CD, et al. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet 1984;1:533-5.
Fertility and Sterility