Vol. 56, No.6, December 1991
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright ~ 1991 The American Fertility Society
Cryopreservation of the occasionally improved semen samples for intrauterine insemination: a new approach in the treatment of idiopathic male infertility
Mohamed A. Aboulghar, M.D.*t:!: Ragaa T. Mansour, M.D.t Gamall. Serour, M.D.t§
Mehany A. Sattar, M.D.t§ Inas Elattar, Ph.D.:\:
The Egyptian IVF·ET Center, Cairo University, and Al-Azhar University, Cairo, Egypt
Objective: To assess the value of treating idiopathic male infertility by intrauterine insemination (lUI) of the occasionally improved cryopreserved semen. Design: Two groups of idiopathic oligospermic patients were chosen at random and treated by lUI using processed fresh semen in group A and the best available cryopreserved semen samples pooled with fresh samples in group B. Setting: Egyptian IVF -ET Centre, Maadi, Cairo, Egypt. Patients, Participants: One hundred fifty infertile couples because of idiopathic oligoasthenospermia. Intervention: Intrauterine insemination. Main Outcome Measure: The pregnancy rate was evaluated after an average of three treatment cycles. Results: The pregnancy rate (PR) was significantly higher in group B when compared with group A. The improvement in the PR was highly significant in the subgroup of patients for whom reasonable semen samples could be collected and cryopreserved. Conclusions: Our study indicates that lUI with fresh semen pooled with cryopreserved occasionally improved semen samples for the treatment of oligoasthenospermia results in an improved PR. Fertil Steril1991;56:1151-5
It has been estimated that 40% of human infertility is wholly or in part because of deficiencies in sperm numbers and/or sperm motility. 1 A large number of infertile men have normal results on physical examination, normal hormonal profiles, and no discernible cause of their infertility. These individuals fall into the category of idiopathic infertility and constitute approximately 40% of all patients with male infertility.2 Many drugs and hormones were used in the treatment of idiopathic oligospermia, yet there is no evidence to support the
effectiveness of these different therapies. 3 The efficacy of intrauterine insemination (lUI) as a treatment of oligospermic or asthenospermic males is debatable.4~
There is a great deal of variation in the semen of the same patient. 7,8 Thus, males diagnosed as having oligospermia may on occasion have normal counts. Pregnancy without therapy, despite low sperm counts or motilities, have been documented. 9 The aim of the present work is to study the value of lUI using the cryopreserved occasionally improved semen samples in the treatment of infertility caused by oligoasthenospermia.
Received January 11, 1991; revised and accepted August 6, 1991-
* Reprint requests: Mohamed Aboulghar, M.D., The Egyptian IVF-ET Center, 85 Maadi Zeraie Road, Maadi, Cairo, Egypt. t The Egyptian IVF-ET Center. :j: Cairo University. § AI-Azhar University. Vol. 56, No.6, December 1991
MATERIALS AND METHODS
One hundred fifty couples whose infertility was thought to be because of oligoasthenospermia were Aboulghar et al.
Cryopreserving occasional improved semen
1151
included in this study. All female partners showed normal findings on clinical examination. Their tubes were patent as shown by hysterosalpingography or laparoscopy. They were regularly ovulating as shown by regular menstruation and serum progesterone. Clinical characteristics are shown in Table 1. The evaluation of semen parameters was done in our laboratory by the same pathologist according to the World Health Organization Laboratory Manual.s Two semen samples were examined for each patient. The semen was considered subfertile when the count was 40%. Patients were chosen with a minimum sperm count of 3 X 106/mL and a maximum of 15 X 106/mL with a minimal motility of 30%. The duration of infertility ranged from 2 to 15 years. All cases were primary infertility. The patients were divided into two groups at random. Seventy-seven patients (group A) were treated by our standard protocol of superovulation and lUI. All patients received clomiphene citrate (Clomid; Merrel-Dow- France S.A., Neuilly-sur- Seine, France) 100 to 150 mg/d on days 3 to 7 of the cycle and human menopausal gonadotropin (hMG, (Pergonal; I.F. Serono S.P.A., Rome, Italy) one to three ampules per day starting on day 6 of the cycle. The dose was modified in subsequent cycles, depending on the patient's response. Daily vaginal ultrasound for monitoring of ovarian response started on day 8. Measurements of luteinizing hormone in urine (monocolonal antibodies, catalog No. 02-094: Monoclonal Antibodies, Inc., Sunnyvale, CA) was done every 8 hours starting from day 10 of the cycle. The basic protocol was to administer hMG until the leading follicle reached a mean diameter of 17 mm. Human chorionic gonadotropin was not given to trigger ovulation, and lUI was timed 24 hours after the detection of the peak of the LH surge in the urine. On the day of insemination, the semen sample was collected in a sterile container by masturbation. After liquefaction at room temperature (RT), assessment of count and motility was done. The whole semen was allocated into two centrifuge tubes and Table 1
o
Female Patients Characteristics Characteristic
Group A
No. of patients Age (y)O Duration of infertility (y)O
77
73
33.2 ± 5.4 (24 to 38) 6.3 ± 2.9 (3 to 14)
34.1 ± 4.9 (25 to 39) 6.5 ± 3.1 (2 to 15)
Values are means ± SD with ranges in parentheses.
1152
combined with Ham's F-10 media (GIBCO Laboratory, Grand Island, NY) in 1:1 ratio. If the semen sample was highly viscous, additional media were added, and uniform mixing using an 18-G needle was obtained. Tubes were centrifuged at 1,000 rpm for 7 minutes. The supernatant was discarded, the pellet was resuspended in 1 mL of media, and the process was repeated. After this final wash, 1 mL of Ham's F-10 media supplemented with 7.5% heatinactivated serum was gently layered over the undisturbed pellet, and the tubes were incubated for 1 hour at 37°C and 5% CO 2 in air. The supernatant was aspirated into the insemination catheter using a 1-cc syringe. Insemination was repeated for an average of 2.8 cycles per patient within 1 year (ranging from 2 to 4 cycles). Seventy-three patients (group B) were followed up by a new semen analysis every 2 to 4 weeks for a period of 8 months until one or more occasional reasonable samples were collected and cryopreserved. The semen sample was considered reasonable if the count was ~15 X 106/mL, the motility was ~35%, and abnormal forms were 0.05.
Fertility and Sterility
Table 2
Composition of Cryoprotectant Medium a
100
Weight
Material
80 g
NaCI KCl Ca Lactate MgSO. Na H2 PO. 2H 20 Na HCO g Hepes Glucose Fructose Streptomycin Penicillin Human serum albumin Glycin
Q)
en
5.80 0.40 0.76 0.12 0.05 2.60 4.77 8.59 8.59 0.05 0.05 4.00 10.00
260
c
Q)
o
81
~ 40
a.. 20
, .....
82
............
A
0+------,-----1'·-'-""0.;·'"'-'.'.'=.'"'-"'='=''-'-'T-! ' ------,
o
123
4
Number of cycles
The above was dissolved in 850 mL distilled H 20, and 150 mL of glycerol was added. a
as in group A. Insemination was done using all the available motile sperms from the fresh and cryopreserved samples. The procedure was repeated for an average of three cycles, ranging from 2 to 4 cycles within 1 year. The statistical methods used in the analysis were Student's t-test to compare groups A and B with respect to age and duration of infertility. Cumulative pregnancy rates (PR) were calculated using the life table analysis, and statistical comparison between the curves was performed using generalized Wilcoxon's test.lO Probability value < 0.05 was considered significant.
RESULTS One hundred fifty couples underwent a total of 428 treatment cycles of lUI. They were divided into two groups. Group A (n = 77) underwent 219 treatment cycles (2.8 ± 0.54 cycles/patient). Pregnancy occurred in 9 cases, resulting in an average PR of 4.1 % per cycle and a cumulative PR of 13% per couple after 3 treatment cycles. Group B (n = 73) underwent 209 treatment cycles (2.9 ± 0.52 cycles/pa-
Figure 1 Cumulative PRs of patients in groups A, B1, and B2 (P = 0.024 using generalized Wilcoxon's test).
tient). Pregnancy occurred in 19 cases, resulting in an average PR of 9.1 % per cycle and a cumulative PR of 27.7%. The cumulative pregnancy curve of patients in group A was significantly different from those in group B, P = 0.027. Group B was divided into two subgroups: subgroup Bl (n = 36) in which reasonable samples were collected and used for lUI resulting in 13 pregnancies (average PR 12.3% per cycle and cumulative PR 36% per couple) and subgroup B2 (n = 37) in which a reasonable sample could not be collected, and lUI was done using the best possible semen samples. Pregnancy occurred in six cases with an average PR 5.8% per cycle and cumulative PR 17.7% per couple (Table 3). Pregnancy rate in subgroup Bl was significantly higher than in group A; there was no significant difference in PR between subgroup B2 and group A (P = 0.024). The cumulative PRs of the three groups are shown in Figure l. The average number of semen analysis in group B was 5 and the average period until a reasonable sample was collected was 4 months (range of 2 to 8 months). Semen characteristics of all groups are
Table 3
Results of Treatment in All Groups
Groups
No. of couples
No. of cycles
No. of pregnancies
Cumulative PR/couple a
Group A Group B, Group B2
77 36 37
219 106 103
9 13 6
13.0 36.0 17.7
Average PR/cycle %
4.1 12.3 5.8
a Because a very small number of patients received four treatment cycles (6 in group A and 5 in group B), the cumulative percentage of patients achieving pregnancy after three treatment cycles using life table method is reported.
Vol. 56, No.6, December 1991
Aboulghar et al.
Cryopreserving occasional improved semen
1153
shown in Table 4. The average total motile sperms used for lUI was significantly higher in group B when compared with group A and group B1 when compared with group B2 (P < 0.05). After cryopreservation and thawing, only 40% of the total motile sperms were viable and were used for lUI. The minimal total number of motile sperms that resulted in pregnancy after lUI was 2.9 X 106. During the period ofthe study, three independent pregnancies occurred in cycles with no lUI, and these pregnancies were not included in our results. DISCUSSION
It was clearly demonstrated that women attending an infertility clinic can conceive, although their partners have markedly abnormal semen profiles. l l The marked variability of sperm output and quality that occurs spontaneously may result in pregnancies independent of treatment. 9 ,12 In the present study, PR was significantly higher in group B when compared with group A. This could be explained by the fact that the total number of motile sperms used for insemination was significantly higher in group B when compared with group A. In group B, one or more cryopreserved samples after thawing and one fresh sample were pooled together and used for lUI. Repeated semen analysis over a period of several months enabled us to collect better semen quality samples in 49% of the patients (subgroup B1). It was reported that lUI with 10 X 106 total motile sperm. In oligospermic men, theoretically speaking, the possibilities of having an improved semen sample exactly at the time of ovulation and the occurrence of intercourse at the right time is probably remote. Cryopreserving the occasional improved semen samples and its use for lUI at the time of ovulation seems to maximize the chances of pregnancy in these patients. Superovulation will also increase the efficacy of IUI. 14 It was shown from our study that 49% of group B patients showed spontaneous improvement in semen samples during the follow-up period. This subgroup of patients (B1) showed a highly significant improved PR when compared with patients who did not show the improvement (B2). This could be explained by the high number of total motile sperms collected during the improvement period and used for lUI. Group B1 includes probably a number of patients who eventually achieve spontaneous pregnancy after a long period of infertility because of male factor. This further supports the work of Tur-Kaspa et al.,15 who reported a better PR by pooling fresh sequentional samples to be used for lUI. It has been repeatedly reported that crypreservation negatively influences sperm motility and longevity and disrupts acrosomal membranes. 16,17 This study showed that the motile sperms decreased by 60%, but the forward grade of the sperms that survived was not affected. In our experience, the loss of motile sperm is only 30% in normal semen collected from fertile men using the same cryoprotec-
Semen Characteristics and Total Sperm Counts Used for lUI in Both Groups Total motile sperm Total count
Motile count
After thawing
%
XIO'
XIO'
30 ± 9.5
5.3 ± 2.5
Motility
Used for lUI
Groups
Count
XIO'
Group A Group B Average basal Average improved samples Bl (average improved) B2 (average best available)
8.5 ± 3.5"
22 ± 6.5
9.1 ± 4.1
23 ± 7.1
32 ± 10.4
21.0 ± 8.1
50 ± 12.0
41 ± 15.3
20.0 ± 8.0
8± 2.9
19.0 ± 7.1 b
32.0 ± 11.0
78 ± 16.0
51 ± 16.0
39.0 ± 12.0
16 ± 3.5
36.0 ± 12.0 b
12.0 ± 3.4
29 ± 9.2
32 ± 11.0
10.0 ± 4.0
4 ± 1.2
11.0 ± 4.8 b
XlO'/mL
"Values are means ± SD. 1154
Aboulghar et al.
b
Cryopreserving occasional improved semen
XIO'
5.3 ± 2.5
Two frozen semen samples and one fresh sample.
Fertility and Sterility
tant and the same technique for cryopreservation and thawing for subfertile samples. Byrd et al. 18 studied sperm parameters before and after thawing. They showed no difference in the characteristics of sperms and a significant drop in the average number of motile sperm used for lUI. The use of frozen sperms has been proven to be efficient in IVF .19,20 Englert et al. 21 showed that sperm characteristics before and after preparation for IVF are significantly worse with frozen-thawed than with fresh sperm but without entailing significant impairment of fertilization rates. Morshedi et al. 22 showed that motility of cryopreserved samples after swim-up preparation and insemination maintain a fair motility after 16 hours. They showed that for certain patients with subfertile sperm concentrations and motility, cryopreservation is a viable option with an acceptable prognosis. Although the fertilization rate was diminished in these cases, the overall PRs were comparable with those of frozenthawed donor samples and those of fresh samples from infertile patients having the same semen profile. In conclusion, it is believed that treatment of infertility because of male factor by cryopreserving the occasional improved semen samples and using it for lUI, together with superovulation and close monitoring of the patient, will result in an improved PR. This simple and relatively noninvasive procedure may be tried as a first line of treatment before resorting to more complex and invasive procedures as IVF and gamete intrafallopian transfer.
REFERENCES 1. Speroff L, Glass RH, Kase NG: Male infertility. In Clinical Gynecologic Endocrinology and Infertility, 4th edition, Edited by L Speroff, RH Glass, NG Kase. Baltimore, Williams and Wilkins Co., 1978, p 5652 2. McClure RD: Drug therapy for idiopathic infertility. In Contemporary Management of Impotence and Infertility, Edited by EA Tanagho, TF Lue, RD McClure. Baltimore; Williams and Wilkins Co., 1988, p 291 3. Glass RH, Ericsson RJ: Intrauterine insemination of isolated motile sperm. Fertil Steril 29:535, 1978 4. Horvath PM, Bohrer M, Shelden RM, Kemmann E: The relationship of sperm parameters to cycle fecundity in superovulated women undergoing intrauterine insemination. Fertil Steril 52:288, 1989 5. Kerin J, Quinn P: Washed intrauterine insemination in the treatment of oligospermic infertility. Sem Reprod Endocrinol 5:23,1987 6. Moghissi KS: Some reflections on intrauterine insemination. Fertil Steril 46:13, 1986
Vol. 56, No.6, December 1991
7. Sherins RJ, Brightwell C, Sternthal PM: Longitudinal analysis of semen of fertile and infertile men. In The Testis in Normal and Infertile Men, Edited by P Troen, HR Nankin. New York, Raven Press, 1977, p 473 8. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 28 9. Glass RH, Ericsson RJ: Spontaneous cure of male infertility. Fertil Steril 31:305, 1979 10. Lee LT: Nonparametric methods for estimating survival functions. In Statistical Methods For Survival Data Analysis, Edited by CA Belmont. Lifetime Learning Publications, 1980, p 76 11. Van Zyl JA, Menkveld R, Kotze TJ, van W Retief AE, Niekerk WA: Oligospermia: a seven-year survey of the incidence, chromosomal aberrations, treatment and pregnancy rate. Int J Fertil20:129, 1975 12. Collins JA, Wrixon W, Janes LB, Wilson EH: Treatmentindependent pregnancy among infertile couples. N Engl J Med 309:1201, 1983 13. Bohrer M, Kemmann E, Pasquale S, Shelden RM: The significance of the total number of motile sperm delivered with intrauterine insemination (lUI) in menotropin treated women. (Abstr. P-159) Presented at the 42nd Annual Meeting of The American Fertility Society and the 18th Annual Meeting of the Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 27 to October 2, 1986. Published by The American Fertility Society, in the Program Supplement, 1986, p 56 14. Chaffkin LM, Nulsen JC, Luciano AA, Metzger DA: A comparative analysis of the cycle fecundity rates associated with combined human menopausal gonadotropin (hMG) and intrauterine insemination (lUI) versus either hMG or lUI alone. Fertil Steril 55:252, 1991 15. Tur-Kaspa I, Dudkiewicz A, Confino E, Gleicher N: Pooled sequentional ejaculates: a way to increase the total number of motile sperm from oligozoospermic men. Fertil Steril 54: 906, 1990 16. Steinburger E, Smith KD: Artificial insemination with fresh or frozen semen: a comparative study. JAMA 223:778, 1973 17. Keel BA, Webster BW, Roberts DK: Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil 81:213, 1987 18. Byrd W, Bradshaw K, Carr B, Edman C, Odom J, Ackerman G: A prospective randomized study of pregnancy rates following intrauterine and intracervical insemination using frozen donor sperm. Fertil Steril 53:521, 1990 19. Mahadevan M, Trounson AO, Leeton JF: Successful use of human semen cryobanking for in vitro fertilization. Fertil Steril 40:340, 1983 20. Cohen J, Edwards RG, Fehilly CB, Fishel SB, Hewitt J, Rowland GF, Steptoe PC, Walters DE, Webster J: In vitro fertilization using cryopreserved donor semen in cases where both partners are infertile. Fertil Steril 43:570, 1985 21. Englert Y, Delvigne A, Vekemans M, Lejeune B, Henlisz A, de Maertelaer G, Leroy F: Is fresh or frozen semen to be used in in vitro fertilization with donor sperm? Fertil Steril 51: 661, 1989 22. Morshedi M, Oehninger S, Veeck LL, Ertunc H, Bocca S, Acosta AA: Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients. Fertil Steril 54:1093, 1990
AbouIghar et al.
Cryopreserving occasional improved semen
1155
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright ~ 1991 The American Fertility Society
Cryopreservation of the occasionally improved semen samples for intrauterine insemination: a new approach in the treatment of idiopathic male infertility
Mohamed A. Aboulghar, M.D.*t:!: Ragaa T. Mansour, M.D.t Gamall. Serour, M.D.t§
Mehany A. Sattar, M.D.t§ Inas Elattar, Ph.D.:\:
The Egyptian IVF·ET Center, Cairo University, and Al-Azhar University, Cairo, Egypt
Objective: To assess the value of treating idiopathic male infertility by intrauterine insemination (lUI) of the occasionally improved cryopreserved semen. Design: Two groups of idiopathic oligospermic patients were chosen at random and treated by lUI using processed fresh semen in group A and the best available cryopreserved semen samples pooled with fresh samples in group B. Setting: Egyptian IVF -ET Centre, Maadi, Cairo, Egypt. Patients, Participants: One hundred fifty infertile couples because of idiopathic oligoasthenospermia. Intervention: Intrauterine insemination. Main Outcome Measure: The pregnancy rate was evaluated after an average of three treatment cycles. Results: The pregnancy rate (PR) was significantly higher in group B when compared with group A. The improvement in the PR was highly significant in the subgroup of patients for whom reasonable semen samples could be collected and cryopreserved. Conclusions: Our study indicates that lUI with fresh semen pooled with cryopreserved occasionally improved semen samples for the treatment of oligoasthenospermia results in an improved PR. Fertil Steril1991;56:1151-5
It has been estimated that 40% of human infertility is wholly or in part because of deficiencies in sperm numbers and/or sperm motility. 1 A large number of infertile men have normal results on physical examination, normal hormonal profiles, and no discernible cause of their infertility. These individuals fall into the category of idiopathic infertility and constitute approximately 40% of all patients with male infertility.2 Many drugs and hormones were used in the treatment of idiopathic oligospermia, yet there is no evidence to support the
effectiveness of these different therapies. 3 The efficacy of intrauterine insemination (lUI) as a treatment of oligospermic or asthenospermic males is debatable.4~
There is a great deal of variation in the semen of the same patient. 7,8 Thus, males diagnosed as having oligospermia may on occasion have normal counts. Pregnancy without therapy, despite low sperm counts or motilities, have been documented. 9 The aim of the present work is to study the value of lUI using the cryopreserved occasionally improved semen samples in the treatment of infertility caused by oligoasthenospermia.
Received January 11, 1991; revised and accepted August 6, 1991-
* Reprint requests: Mohamed Aboulghar, M.D., The Egyptian IVF-ET Center, 85 Maadi Zeraie Road, Maadi, Cairo, Egypt. t The Egyptian IVF-ET Center. :j: Cairo University. § AI-Azhar University. Vol. 56, No.6, December 1991
MATERIALS AND METHODS
One hundred fifty couples whose infertility was thought to be because of oligoasthenospermia were Aboulghar et al.
Cryopreserving occasional improved semen
1151
included in this study. All female partners showed normal findings on clinical examination. Their tubes were patent as shown by hysterosalpingography or laparoscopy. They were regularly ovulating as shown by regular menstruation and serum progesterone. Clinical characteristics are shown in Table 1. The evaluation of semen parameters was done in our laboratory by the same pathologist according to the World Health Organization Laboratory Manual.s Two semen samples were examined for each patient. The semen was considered subfertile when the count was 40%. Patients were chosen with a minimum sperm count of 3 X 106/mL and a maximum of 15 X 106/mL with a minimal motility of 30%. The duration of infertility ranged from 2 to 15 years. All cases were primary infertility. The patients were divided into two groups at random. Seventy-seven patients (group A) were treated by our standard protocol of superovulation and lUI. All patients received clomiphene citrate (Clomid; Merrel-Dow- France S.A., Neuilly-sur- Seine, France) 100 to 150 mg/d on days 3 to 7 of the cycle and human menopausal gonadotropin (hMG, (Pergonal; I.F. Serono S.P.A., Rome, Italy) one to three ampules per day starting on day 6 of the cycle. The dose was modified in subsequent cycles, depending on the patient's response. Daily vaginal ultrasound for monitoring of ovarian response started on day 8. Measurements of luteinizing hormone in urine (monocolonal antibodies, catalog No. 02-094: Monoclonal Antibodies, Inc., Sunnyvale, CA) was done every 8 hours starting from day 10 of the cycle. The basic protocol was to administer hMG until the leading follicle reached a mean diameter of 17 mm. Human chorionic gonadotropin was not given to trigger ovulation, and lUI was timed 24 hours after the detection of the peak of the LH surge in the urine. On the day of insemination, the semen sample was collected in a sterile container by masturbation. After liquefaction at room temperature (RT), assessment of count and motility was done. The whole semen was allocated into two centrifuge tubes and Table 1
o
Female Patients Characteristics Characteristic
Group A
No. of patients Age (y)O Duration of infertility (y)O
77
73
33.2 ± 5.4 (24 to 38) 6.3 ± 2.9 (3 to 14)
34.1 ± 4.9 (25 to 39) 6.5 ± 3.1 (2 to 15)
Values are means ± SD with ranges in parentheses.
1152
combined with Ham's F-10 media (GIBCO Laboratory, Grand Island, NY) in 1:1 ratio. If the semen sample was highly viscous, additional media were added, and uniform mixing using an 18-G needle was obtained. Tubes were centrifuged at 1,000 rpm for 7 minutes. The supernatant was discarded, the pellet was resuspended in 1 mL of media, and the process was repeated. After this final wash, 1 mL of Ham's F-10 media supplemented with 7.5% heatinactivated serum was gently layered over the undisturbed pellet, and the tubes were incubated for 1 hour at 37°C and 5% CO 2 in air. The supernatant was aspirated into the insemination catheter using a 1-cc syringe. Insemination was repeated for an average of 2.8 cycles per patient within 1 year (ranging from 2 to 4 cycles). Seventy-three patients (group B) were followed up by a new semen analysis every 2 to 4 weeks for a period of 8 months until one or more occasional reasonable samples were collected and cryopreserved. The semen sample was considered reasonable if the count was ~15 X 106/mL, the motility was ~35%, and abnormal forms were 0.05.
Fertility and Sterility
Table 2
Composition of Cryoprotectant Medium a
100
Weight
Material
80 g
NaCI KCl Ca Lactate MgSO. Na H2 PO. 2H 20 Na HCO g Hepes Glucose Fructose Streptomycin Penicillin Human serum albumin Glycin
Q)
en
5.80 0.40 0.76 0.12 0.05 2.60 4.77 8.59 8.59 0.05 0.05 4.00 10.00
260
c
Q)
o
81
~ 40
a.. 20
, .....
82
............
A
0+------,-----1'·-'-""0.;·'"'-'.'.'=.'"'-"'='=''-'-'T-! ' ------,
o
123
4
Number of cycles
The above was dissolved in 850 mL distilled H 20, and 150 mL of glycerol was added. a
as in group A. Insemination was done using all the available motile sperms from the fresh and cryopreserved samples. The procedure was repeated for an average of three cycles, ranging from 2 to 4 cycles within 1 year. The statistical methods used in the analysis were Student's t-test to compare groups A and B with respect to age and duration of infertility. Cumulative pregnancy rates (PR) were calculated using the life table analysis, and statistical comparison between the curves was performed using generalized Wilcoxon's test.lO Probability value < 0.05 was considered significant.
RESULTS One hundred fifty couples underwent a total of 428 treatment cycles of lUI. They were divided into two groups. Group A (n = 77) underwent 219 treatment cycles (2.8 ± 0.54 cycles/patient). Pregnancy occurred in 9 cases, resulting in an average PR of 4.1 % per cycle and a cumulative PR of 13% per couple after 3 treatment cycles. Group B (n = 73) underwent 209 treatment cycles (2.9 ± 0.52 cycles/pa-
Figure 1 Cumulative PRs of patients in groups A, B1, and B2 (P = 0.024 using generalized Wilcoxon's test).
tient). Pregnancy occurred in 19 cases, resulting in an average PR of 9.1 % per cycle and a cumulative PR of 27.7%. The cumulative pregnancy curve of patients in group A was significantly different from those in group B, P = 0.027. Group B was divided into two subgroups: subgroup Bl (n = 36) in which reasonable samples were collected and used for lUI resulting in 13 pregnancies (average PR 12.3% per cycle and cumulative PR 36% per couple) and subgroup B2 (n = 37) in which a reasonable sample could not be collected, and lUI was done using the best possible semen samples. Pregnancy occurred in six cases with an average PR 5.8% per cycle and cumulative PR 17.7% per couple (Table 3). Pregnancy rate in subgroup Bl was significantly higher than in group A; there was no significant difference in PR between subgroup B2 and group A (P = 0.024). The cumulative PRs of the three groups are shown in Figure l. The average number of semen analysis in group B was 5 and the average period until a reasonable sample was collected was 4 months (range of 2 to 8 months). Semen characteristics of all groups are
Table 3
Results of Treatment in All Groups
Groups
No. of couples
No. of cycles
No. of pregnancies
Cumulative PR/couple a
Group A Group B, Group B2
77 36 37
219 106 103
9 13 6
13.0 36.0 17.7
Average PR/cycle %
4.1 12.3 5.8
a Because a very small number of patients received four treatment cycles (6 in group A and 5 in group B), the cumulative percentage of patients achieving pregnancy after three treatment cycles using life table method is reported.
Vol. 56, No.6, December 1991
Aboulghar et al.
Cryopreserving occasional improved semen
1153
shown in Table 4. The average total motile sperms used for lUI was significantly higher in group B when compared with group A and group B1 when compared with group B2 (P < 0.05). After cryopreservation and thawing, only 40% of the total motile sperms were viable and were used for lUI. The minimal total number of motile sperms that resulted in pregnancy after lUI was 2.9 X 106. During the period ofthe study, three independent pregnancies occurred in cycles with no lUI, and these pregnancies were not included in our results. DISCUSSION
It was clearly demonstrated that women attending an infertility clinic can conceive, although their partners have markedly abnormal semen profiles. l l The marked variability of sperm output and quality that occurs spontaneously may result in pregnancies independent of treatment. 9 ,12 In the present study, PR was significantly higher in group B when compared with group A. This could be explained by the fact that the total number of motile sperms used for insemination was significantly higher in group B when compared with group A. In group B, one or more cryopreserved samples after thawing and one fresh sample were pooled together and used for lUI. Repeated semen analysis over a period of several months enabled us to collect better semen quality samples in 49% of the patients (subgroup B1). It was reported that lUI with 10 X 106 total motile sperm. In oligospermic men, theoretically speaking, the possibilities of having an improved semen sample exactly at the time of ovulation and the occurrence of intercourse at the right time is probably remote. Cryopreserving the occasional improved semen samples and its use for lUI at the time of ovulation seems to maximize the chances of pregnancy in these patients. Superovulation will also increase the efficacy of IUI. 14 It was shown from our study that 49% of group B patients showed spontaneous improvement in semen samples during the follow-up period. This subgroup of patients (B1) showed a highly significant improved PR when compared with patients who did not show the improvement (B2). This could be explained by the high number of total motile sperms collected during the improvement period and used for lUI. Group B1 includes probably a number of patients who eventually achieve spontaneous pregnancy after a long period of infertility because of male factor. This further supports the work of Tur-Kaspa et al.,15 who reported a better PR by pooling fresh sequentional samples to be used for lUI. It has been repeatedly reported that crypreservation negatively influences sperm motility and longevity and disrupts acrosomal membranes. 16,17 This study showed that the motile sperms decreased by 60%, but the forward grade of the sperms that survived was not affected. In our experience, the loss of motile sperm is only 30% in normal semen collected from fertile men using the same cryoprotec-
Semen Characteristics and Total Sperm Counts Used for lUI in Both Groups Total motile sperm Total count
Motile count
After thawing
%
XIO'
XIO'
30 ± 9.5
5.3 ± 2.5
Motility
Used for lUI
Groups
Count
XIO'
Group A Group B Average basal Average improved samples Bl (average improved) B2 (average best available)
8.5 ± 3.5"
22 ± 6.5
9.1 ± 4.1
23 ± 7.1
32 ± 10.4
21.0 ± 8.1
50 ± 12.0
41 ± 15.3
20.0 ± 8.0
8± 2.9
19.0 ± 7.1 b
32.0 ± 11.0
78 ± 16.0
51 ± 16.0
39.0 ± 12.0
16 ± 3.5
36.0 ± 12.0 b
12.0 ± 3.4
29 ± 9.2
32 ± 11.0
10.0 ± 4.0
4 ± 1.2
11.0 ± 4.8 b
XlO'/mL
"Values are means ± SD. 1154
Aboulghar et al.
b
Cryopreserving occasional improved semen
XIO'
5.3 ± 2.5
Two frozen semen samples and one fresh sample.
Fertility and Sterility
tant and the same technique for cryopreservation and thawing for subfertile samples. Byrd et al. 18 studied sperm parameters before and after thawing. They showed no difference in the characteristics of sperms and a significant drop in the average number of motile sperm used for lUI. The use of frozen sperms has been proven to be efficient in IVF .19,20 Englert et al. 21 showed that sperm characteristics before and after preparation for IVF are significantly worse with frozen-thawed than with fresh sperm but without entailing significant impairment of fertilization rates. Morshedi et al. 22 showed that motility of cryopreserved samples after swim-up preparation and insemination maintain a fair motility after 16 hours. They showed that for certain patients with subfertile sperm concentrations and motility, cryopreservation is a viable option with an acceptable prognosis. Although the fertilization rate was diminished in these cases, the overall PRs were comparable with those of frozenthawed donor samples and those of fresh samples from infertile patients having the same semen profile. In conclusion, it is believed that treatment of infertility because of male factor by cryopreserving the occasional improved semen samples and using it for lUI, together with superovulation and close monitoring of the patient, will result in an improved PR. This simple and relatively noninvasive procedure may be tried as a first line of treatment before resorting to more complex and invasive procedures as IVF and gamete intrafallopian transfer.
REFERENCES 1. Speroff L, Glass RH, Kase NG: Male infertility. In Clinical Gynecologic Endocrinology and Infertility, 4th edition, Edited by L Speroff, RH Glass, NG Kase. Baltimore, Williams and Wilkins Co., 1978, p 5652 2. McClure RD: Drug therapy for idiopathic infertility. In Contemporary Management of Impotence and Infertility, Edited by EA Tanagho, TF Lue, RD McClure. Baltimore; Williams and Wilkins Co., 1988, p 291 3. Glass RH, Ericsson RJ: Intrauterine insemination of isolated motile sperm. Fertil Steril 29:535, 1978 4. Horvath PM, Bohrer M, Shelden RM, Kemmann E: The relationship of sperm parameters to cycle fecundity in superovulated women undergoing intrauterine insemination. Fertil Steril 52:288, 1989 5. Kerin J, Quinn P: Washed intrauterine insemination in the treatment of oligospermic infertility. Sem Reprod Endocrinol 5:23,1987 6. Moghissi KS: Some reflections on intrauterine insemination. Fertil Steril 46:13, 1986
Vol. 56, No.6, December 1991
7. Sherins RJ, Brightwell C, Sternthal PM: Longitudinal analysis of semen of fertile and infertile men. In The Testis in Normal and Infertile Men, Edited by P Troen, HR Nankin. New York, Raven Press, 1977, p 473 8. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 28 9. Glass RH, Ericsson RJ: Spontaneous cure of male infertility. Fertil Steril 31:305, 1979 10. Lee LT: Nonparametric methods for estimating survival functions. In Statistical Methods For Survival Data Analysis, Edited by CA Belmont. Lifetime Learning Publications, 1980, p 76 11. Van Zyl JA, Menkveld R, Kotze TJ, van W Retief AE, Niekerk WA: Oligospermia: a seven-year survey of the incidence, chromosomal aberrations, treatment and pregnancy rate. Int J Fertil20:129, 1975 12. Collins JA, Wrixon W, Janes LB, Wilson EH: Treatmentindependent pregnancy among infertile couples. N Engl J Med 309:1201, 1983 13. Bohrer M, Kemmann E, Pasquale S, Shelden RM: The significance of the total number of motile sperm delivered with intrauterine insemination (lUI) in menotropin treated women. (Abstr. P-159) Presented at the 42nd Annual Meeting of The American Fertility Society and the 18th Annual Meeting of the Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 27 to October 2, 1986. Published by The American Fertility Society, in the Program Supplement, 1986, p 56 14. Chaffkin LM, Nulsen JC, Luciano AA, Metzger DA: A comparative analysis of the cycle fecundity rates associated with combined human menopausal gonadotropin (hMG) and intrauterine insemination (lUI) versus either hMG or lUI alone. Fertil Steril 55:252, 1991 15. Tur-Kaspa I, Dudkiewicz A, Confino E, Gleicher N: Pooled sequentional ejaculates: a way to increase the total number of motile sperm from oligozoospermic men. Fertil Steril 54: 906, 1990 16. Steinburger E, Smith KD: Artificial insemination with fresh or frozen semen: a comparative study. JAMA 223:778, 1973 17. Keel BA, Webster BW, Roberts DK: Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil 81:213, 1987 18. Byrd W, Bradshaw K, Carr B, Edman C, Odom J, Ackerman G: A prospective randomized study of pregnancy rates following intrauterine and intracervical insemination using frozen donor sperm. Fertil Steril 53:521, 1990 19. Mahadevan M, Trounson AO, Leeton JF: Successful use of human semen cryobanking for in vitro fertilization. Fertil Steril 40:340, 1983 20. Cohen J, Edwards RG, Fehilly CB, Fishel SB, Hewitt J, Rowland GF, Steptoe PC, Walters DE, Webster J: In vitro fertilization using cryopreserved donor semen in cases where both partners are infertile. Fertil Steril 43:570, 1985 21. Englert Y, Delvigne A, Vekemans M, Lejeune B, Henlisz A, de Maertelaer G, Leroy F: Is fresh or frozen semen to be used in in vitro fertilization with donor sperm? Fertil Steril 51: 661, 1989 22. Morshedi M, Oehninger S, Veeck LL, Ertunc H, Bocca S, Acosta AA: Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients. Fertil Steril 54:1093, 1990
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