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Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm
Vol. 58, No.1, July 1992
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright If) 1992 The American Fertility Society
Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm
Y ona Barak, M.Sc. * Ami Amit, M.D. Joseph B. Lessing, M.D.
Gedalia Paz, Ph.D. Zwi T. Homonnai, M.D. Leah Yogev, Ph.D.
In Vitro Fertilization/Embryo Transfer Unit, Tel Aviv Sourasky Medical Center, Serlin Maternity Hospital, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Over the past few years, it has become apparent that male infertility is not entirely reflected by traditional semen analysis. Thus, the absolute values of properties such as concentration, motility, and morphology of spermatozoa are not necessarily related to the fertilizability of the sperm. This conclusion became evident when high fertilization and pregnancy rates (PRs) were achieved by using thawed donor sperm instead of fresh sperm, in spite of the damage that was caused to sperm by the freezing process (1). Donor sperm cryopreservation was performed using medium containing egg yolk (EY). Egg yolk medium was found to enhance the percentage of spermatozoa that underwent the acrosome reaction and sperm penetration of zona-free hamster oocytes. In this study, therefore, the possibility of whether EY might improve the fertilizability of sperm with a low fertilization rate was examined in our in vitro fertilization (IVF) program. MATERIALS AND METHODS
In this investigation, the women underwent controlled ovarian hyperstimulation with human menopausal gonadotropin and human chorionic gonadotropin as is done routinely in our unit. The study included two groups of patients. Group A
Received November 1, 1991; revised and accepted March 3, 1992. * Reprint requests: Yona Barak, M.Sc., IVF Unit, Serlin Maternity Hospital, Post Office Box 7079, Tel Aviv 61070, Israel.
Vol. 58, No.1, July 1992
(controls) included 13 couples who underwent 13 IVF cycles; all of them had had one or more previous IVF cycles with a fertilization rate of ~50% in each cycle (79% ± 4.9%, mean ± SE). Group B (the study group) included 19 couples who had undergone 20 IVF cycles. Four of the 19 couples had undergone 1 previous cycle, and the other 15 couples between 2 and 7 cycles. All the couples had had at least one previous cycle with a fertilization rate of <30% (25% ± 4.9%, mean ± SE). Each freshly obtained semen specimen was divided into two equal aliquots. One served as the control and was processed routinely using the double-step washing technique, with insemination medium composed of Ham's F-lO medium (Flow Laboratories, Irvine, Scotland) with 10% patient serum. The other was also initially washed with insemination medium, and the pellet was resuspended with 1.0 mL of EY refrigerating medium (Irvine Scientific, Santa Ana, CA) and was incubated for 2 hours at room temperature. Thereafter 2 mL of insemination medium was added. After centrifugation, the pellet of each part was resuspended in 0.5 mL of insemination medium. As far as maturation of the ova was concerned, aspirated ova from each patient were divided into two equal groups. The ova were inseminated at the same time, either by the control insemination medium sperm or by the EY -treated sperm, 1 to 3 hours after aspiration was completed. The presence of the two pronuclei (2PN) stage was checked 16 to 18 hours after insemination. Differences between treatments were tested for significance using the X2 test.
Barak et al.
Communications-in-brief
197
RESULTS
In group A, selected for couples who showed a previously high fertilization rate, no difference in fertilization rate was found between the oocytes inseminated with sperm washed by the routine IVF procedure with insemination medium and the 00cytes inseminated by sperm pre incubated in EY buffer (Table 1). However, a significant increase (P < 0.01) in the fertilization rate was achieved in oocytes inseminated with sperm pretreated with EY buffer when the couples were selected for a previously low fertilization rate (group B).
DISCUSSION
The effectiveness of EY for sperm preservation at low temperature has been known for years. However, the mechanism by which it provides protection against cold shock is not yet clear. It has been suggested that stabilization of sperm membrane and/or maintenance of the colloid pressure of the external medium might be affected by the EY activity (2). Furthermore, it has been suggested that EY contributes two distinct factors, one of which protects against cold shock (a resistance factor), and a second maintains viability (a storage factor). A low-density lipoprotein fraction is well established as an active constituent in storage and during freezing and thawing. This ingredient with phospholipids might exert the protective action during cold shock, freezing, and cold storage (3). In the present study, it was shown that EY has an ability to improve the fertilizing capacity of sperm selected for a low fertilization rate in previous IVF cycles. The reason for this improved result is unknown and will not be clarified until further research is performed. It could be that EY stabilizes changes on the sperm membrane. It could also be that if some components of the EY penetrate the sperm cell, they stabilize some internal changes that are necessary for fertilization. Another possibility is that components of the EY reduce the damage of sperm membrane caused by free radicals produced by peroxidase activity. This activity has been shown to take place in the sperm cell during incubation (4). This technique, if applied in couples with predominantly male factor infertility, may improve the results of IVF without the need
198
Barak et al.
Communications-in-brief
Table 1 Fertilization Rate of Oocytes Inseminated by Spermatozoa Pre incubated in Insemination Medium or EY Buffer Control group A Insemination medium * No. of aspirated oocytes No. of fertilized oocytes Fertilization rate (%) No.ofIVF cycles
EYt
Study group B Insemination medium *
EYt
81
65
73
81
66
52
22
42
81.5
80
30.1+
51.9
13
20
* Group of oocytes inseminated by spermatozoa washed in the routine IVF procedure. t Group of oocytes inseminated by spermatozoa pretreated with egg yolk buffer. + Significantly different (P < 0.01) according to x' test.
to resort to sperm microinjection or other techniques. SUMMARY
High fertilization and PRs have been achieved by using thawed donor sperm cryopreserved with medium containing EY. The aim of our study was to examine the possible effect of EY on the fertilizing capacity of fresh sperm in an IVF program. Preincubation of spermatozoa in EY for 2 hours at room temperature significantly improved fertilization (P < 0.001) in couples who had low fertilization rates in previous cycles, whereas no effect was found concerning couples with high fertilization rates. Key Words: Egg yolk, in vitro fertilization, fertilization rate, sperm, sperm preparation.
REFERENCES 1. Yavetz H, Yogev L, Homonnai ZT, Paz G. Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time. Fertil Steril 1991;55:812-6. 2. Foulkes JA. The separation of lipoprotein from egg-yolk and their effect on the motility and the integrity of bovine spermatozoa. J Reprod Fertil 1977;49:277-84. 3. Quinn PJ, Chow PYW, White IG. Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site. J Reprod FertiI1980;60:403-7. 4. Aitken RJ, Irvine DS, Wu, FC. Prospective analysis of spermoocyte fusion and reactive oxygen species generation as criteria for diagnosis of infertility. Am J Obstet Gynecol 1991;164:542-51.
Fertility and Sterility
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright If) 1992 The American Fertility Society
Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm
Y ona Barak, M.Sc. * Ami Amit, M.D. Joseph B. Lessing, M.D.
Gedalia Paz, Ph.D. Zwi T. Homonnai, M.D. Leah Yogev, Ph.D.
In Vitro Fertilization/Embryo Transfer Unit, Tel Aviv Sourasky Medical Center, Serlin Maternity Hospital, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Over the past few years, it has become apparent that male infertility is not entirely reflected by traditional semen analysis. Thus, the absolute values of properties such as concentration, motility, and morphology of spermatozoa are not necessarily related to the fertilizability of the sperm. This conclusion became evident when high fertilization and pregnancy rates (PRs) were achieved by using thawed donor sperm instead of fresh sperm, in spite of the damage that was caused to sperm by the freezing process (1). Donor sperm cryopreservation was performed using medium containing egg yolk (EY). Egg yolk medium was found to enhance the percentage of spermatozoa that underwent the acrosome reaction and sperm penetration of zona-free hamster oocytes. In this study, therefore, the possibility of whether EY might improve the fertilizability of sperm with a low fertilization rate was examined in our in vitro fertilization (IVF) program. MATERIALS AND METHODS
In this investigation, the women underwent controlled ovarian hyperstimulation with human menopausal gonadotropin and human chorionic gonadotropin as is done routinely in our unit. The study included two groups of patients. Group A
Received November 1, 1991; revised and accepted March 3, 1992. * Reprint requests: Yona Barak, M.Sc., IVF Unit, Serlin Maternity Hospital, Post Office Box 7079, Tel Aviv 61070, Israel.
Vol. 58, No.1, July 1992
(controls) included 13 couples who underwent 13 IVF cycles; all of them had had one or more previous IVF cycles with a fertilization rate of ~50% in each cycle (79% ± 4.9%, mean ± SE). Group B (the study group) included 19 couples who had undergone 20 IVF cycles. Four of the 19 couples had undergone 1 previous cycle, and the other 15 couples between 2 and 7 cycles. All the couples had had at least one previous cycle with a fertilization rate of <30% (25% ± 4.9%, mean ± SE). Each freshly obtained semen specimen was divided into two equal aliquots. One served as the control and was processed routinely using the double-step washing technique, with insemination medium composed of Ham's F-lO medium (Flow Laboratories, Irvine, Scotland) with 10% patient serum. The other was also initially washed with insemination medium, and the pellet was resuspended with 1.0 mL of EY refrigerating medium (Irvine Scientific, Santa Ana, CA) and was incubated for 2 hours at room temperature. Thereafter 2 mL of insemination medium was added. After centrifugation, the pellet of each part was resuspended in 0.5 mL of insemination medium. As far as maturation of the ova was concerned, aspirated ova from each patient were divided into two equal groups. The ova were inseminated at the same time, either by the control insemination medium sperm or by the EY -treated sperm, 1 to 3 hours after aspiration was completed. The presence of the two pronuclei (2PN) stage was checked 16 to 18 hours after insemination. Differences between treatments were tested for significance using the X2 test.
Barak et al.
Communications-in-brief
197
RESULTS
In group A, selected for couples who showed a previously high fertilization rate, no difference in fertilization rate was found between the oocytes inseminated with sperm washed by the routine IVF procedure with insemination medium and the 00cytes inseminated by sperm pre incubated in EY buffer (Table 1). However, a significant increase (P < 0.01) in the fertilization rate was achieved in oocytes inseminated with sperm pretreated with EY buffer when the couples were selected for a previously low fertilization rate (group B).
DISCUSSION
The effectiveness of EY for sperm preservation at low temperature has been known for years. However, the mechanism by which it provides protection against cold shock is not yet clear. It has been suggested that stabilization of sperm membrane and/or maintenance of the colloid pressure of the external medium might be affected by the EY activity (2). Furthermore, it has been suggested that EY contributes two distinct factors, one of which protects against cold shock (a resistance factor), and a second maintains viability (a storage factor). A low-density lipoprotein fraction is well established as an active constituent in storage and during freezing and thawing. This ingredient with phospholipids might exert the protective action during cold shock, freezing, and cold storage (3). In the present study, it was shown that EY has an ability to improve the fertilizing capacity of sperm selected for a low fertilization rate in previous IVF cycles. The reason for this improved result is unknown and will not be clarified until further research is performed. It could be that EY stabilizes changes on the sperm membrane. It could also be that if some components of the EY penetrate the sperm cell, they stabilize some internal changes that are necessary for fertilization. Another possibility is that components of the EY reduce the damage of sperm membrane caused by free radicals produced by peroxidase activity. This activity has been shown to take place in the sperm cell during incubation (4). This technique, if applied in couples with predominantly male factor infertility, may improve the results of IVF without the need
198
Barak et al.
Communications-in-brief
Table 1 Fertilization Rate of Oocytes Inseminated by Spermatozoa Pre incubated in Insemination Medium or EY Buffer Control group A Insemination medium * No. of aspirated oocytes No. of fertilized oocytes Fertilization rate (%) No.ofIVF cycles
EYt
Study group B Insemination medium *
EYt
81
65
73
81
66
52
22
42
81.5
80
30.1+
51.9
13
20
* Group of oocytes inseminated by spermatozoa washed in the routine IVF procedure. t Group of oocytes inseminated by spermatozoa pretreated with egg yolk buffer. + Significantly different (P < 0.01) according to x' test.
to resort to sperm microinjection or other techniques. SUMMARY
High fertilization and PRs have been achieved by using thawed donor sperm cryopreserved with medium containing EY. The aim of our study was to examine the possible effect of EY on the fertilizing capacity of fresh sperm in an IVF program. Preincubation of spermatozoa in EY for 2 hours at room temperature significantly improved fertilization (P < 0.001) in couples who had low fertilization rates in previous cycles, whereas no effect was found concerning couples with high fertilization rates. Key Words: Egg yolk, in vitro fertilization, fertilization rate, sperm, sperm preparation.
REFERENCES 1. Yavetz H, Yogev L, Homonnai ZT, Paz G. Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time. Fertil Steril 1991;55:812-6. 2. Foulkes JA. The separation of lipoprotein from egg-yolk and their effect on the motility and the integrity of bovine spermatozoa. J Reprod Fertil 1977;49:277-84. 3. Quinn PJ, Chow PYW, White IG. Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site. J Reprod FertiI1980;60:403-7. 4. Aitken RJ, Irvine DS, Wu, FC. Prospective analysis of spermoocyte fusion and reactive oxygen species generation as criteria for diagnosis of infertility. Am J Obstet Gynecol 1991;164:542-51.
Fertility and Sterility