FERTILITY AND STERILITY Copyright
©
Vol. 58, No.6, December 1992
Printed on acid-free paper in U.S.A.
1992 The American Fertility Society
Fresh and frozen-thawed human sperm bind in a similar pattern to the zona· pellucida in the hemizona assay
Ronni Gamzu, B.Sc. * Leah Yogev, Ph.D.* Haim Yavetz, M.D.*
Zvi T. Homonnai, M.D.* Yehuda Hiss, M.D.t Gedalia Paz, Ph.D.*:j:
Institute for the Study of Fertility, Serlin Maternity Hospital, Tel Aviv Sourasky Medical Centre, Sackler Faculty of Medicine, and L. Greenberg Institute of Forensic Medicine, Tel Aviv, Israel
The successful use of frozen-thawed sperm in assisted reproductive techniques (ARTs) depends primarily on the sperm cryopreservation technique used to attain sperm cells with high fertilization capacity. Numerous investigations on cryopreserved sperm quality present contradictory conclusions. However, it is generally accepted that the freezing process impairs sperm membrane integrity and reduces the number and velocity of motile sperm cells. The in vitro fertilization (IVF) model was reported by some investigators as attaining equal fertilization rates comparing fresh with frozen-thawed sperm cells (1), whereas others described superiority of the fresh samples (2). In this study, the hemizona assay (HZA), reported as a satisfactory predictor for evaluating the fertility potential of human spermatozoa (3), was used to assess the fertility potential of fresh as opposed to frozen-thawed sperm cells of the same ejaculate. MATERIALS AND METHODS Handling of Semen
Semen was delivered from 12 different donors attending the outpatient clinic who were selected by virtue of good freezability and previous pregnancies.
Received May 11, 1992; revised and accepted July 28, 1992.
* Institute for the Study of Fertility, Serlin Maternity Hospital, Tel Aviv Sourasky Medical Centre, Sackler Faculty of Medicine. t L. Greenberg Institute of Forensic Medicine. :j: Reprint requests: Gedalia Paz, Ph.D., Institute for the Study of Fertility, Serlin Maternity Hospital, Hakirya, Post Office Box 7079, Tel Aviv, Israel 61070.
1254
Gamzu et al.
Communications-in-brief
After liquefaction, sperm concentration and motility were measured, and morphology was evaluated by strict criteria (4). An aliquot of 250 ~L of the fresh semen was twice washed with medium (Ham's F-10 medium [Flow Laboratories, Irvine, Scotland] + 1 % human serum albumin). The pellet was overlaid with 0.5 mL of medium and incubated at 37°C for 1 hour to enable swim-up separation. The remaining semen was frozen in an egg yolk (EY) containing medium, as described earlier (5). After 2 hours of freezing, one straw (0.5 mL) was thawed, and the sperm suspension was twice washed with the medium for swim-up separation as described above. After separation, both fresh and frozen-thawed samples were diluted with the medium to achieve a motile sperm concentration of 500,000 sperm cells/mL. Hemizona Assay
Oocytes obtained from the ovarian tissue of young cadavers were stored in a salt solution, following protocols described elsewhere (3). One day before the assay was carried out, the required number of oocytes was removed from the salt solution and rinsed several times in medium. Leitz micromanipulators (Leica, Wetzlar, Germany) were used for cutting the oocyte. Microblade (K-20 1730; Ktena Products, Denville, NJ) was glued to a metal bar that was bent twice at right angles to be oriented perpendicularly to the holding pipette. The cutting procedure then continued identically to that put forward by Burkman et al. (3). On the day of the assay, each half of the hemizonae pair was coincubated for 4 hours at 37°C with either fresh or frozenthawed sperm from the same sample. Hemizonae Fertility and Sterility
were then removed and rinsed with Ham's F-lO medium, and the number oftightly bound spermatozoa on the outer surface of hemizonae was counted using phase-contrast microscopy (X400). Statistical Evaluation
The sperm basic parameters are expressed as means ± SE. Statistical evaluation of the number Df tightly bound sperm in the HZA was made using the paired t-test. The hemizona index (HZ!) was calculated by the equation: (binding of fresh sperm/ binding of frozen sperm) X 100 = HZI. RESULTS
The mean concentration of the 12 original raw semen samples was 159 ± 16.9 X 106/m L. Normal forms were 19% ± 1.6%. The percent of motile sperm of the fresh and frozen-thawed samples were 46% ± 16.9% and 24% ± 3.8%, respectively. This observed superiority of the fresh samples compared with post-thaw samples, in terms of motility, was statistically significant (P < 0.005). The results ofthe HZA comparing fresh with frozen-thawed samples are given in Table 1. The number of sperm cells bound per hemizona of fresh and frozen-thawed sperm samples was similar, as indicated by the paired t-test. A distinct variation in the number of tightly bound sperm cells was observed throughout the assays, which probably derives from the differing characteristics of the zona pellucida (ZP) because of various stages of oocyte maturation. DISCUSSION
The currently widespread use of cryopreserved semen in a variety of ARTs raises the need for reassurance that the freezing-thawing process does not adversely affect the fertilizing capacity of sperm cells. However, it is commonly believed that conception rates are lower for frozen-thawed semen than for fresh semen. Also, some investigators have written about lower fertilization rates with the IVF model (2). The present study compares the binding properties of fresh versus frozen-thawed human sperm to human ZP using 12 different semen specimens from fertile men. Notwithstanding the significant reduction in motility after the freezing-thawing process, the binding capacity of the two treatments did not differ. These results are contradicted by that of Coddington et al. (6), who reported a statistically Vol. 58, No.6, December 1992
Table 1 Difference in the Binding Capacity of Fresh Versus Frozen-Thawed Sperm to the ZP in the HZA No. of sperm tightly bound to hemizonae Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 Mean ± SE
Fresh
Frozen-thawed
HZI-
48 9 14 45 82 128 86 88 88 95 150
62 8 12 38 80 118 82 96 82 88 152 66 74 ± 11.9
77.4 112.5 116.6 118.4 102.5 108.4 104.8 91.6 107.3 107.9 98.6 109.0 104.5 ± 3.2
72
75 ± 12.0
- HZI = (fresh/frozen) X 100.
insignificant reduction of approximately 30% in the binding of frozen-thawed sperm compared with the fresh samples. This discrepancy could be attributed to different freezing media and to the fact that Coddington et al. (6) used two different ejaculates as a source for the fresh and the frozen-thawed samples. It would thus seem that when frozen and thawed in EY containing media, the ability of the retrieved sperm cells to bind to the ZP is fully maintained. These conclusions are limited to high quality sperm specimens (e.g., sperm samples from donors); the response of low quality sperm specimens to this process has yet to be evaluated. Our results also support the possible use of frozenthawed donor human sperm specimens as controls in the HZA procedure. This should enable an assay of greater control that could be put to use as a routine diagnostic tool. SUMMARY
This study evaluated the impact of sperm cryopreservation on sperm quality. The HZA was used to test the binding capacity of fresh as opposed to frozen-thawed sperm from 12 donors. Fresh and frozen-thawed sperm motility was 47% ± 1.5% and 24% ± 3.8% (mean ± SE), respectively. However, the number of sperm cells attached to the hemizonae was 75 ± 12.0 and 74 ± 11.9, respectively. We conclude that cryopreservation results in a reduced number of motile sperm cells but does not adversely affect the ability of rescued sperm cells to bind to Gamzu et al.
Communications-in-brief
1255
the ZP. The study also supports the use of frozenthawed rather than fresh donor sperm for control in the HZA procedure. Key Words: Cryopreservation, zona pellucida, hemizona assay. Acknowledgment. We greatly appreciate the scientific and editorial advising of Dov Lichtenberg, Ph.D., Physiology and Pharmacology Department, Sackler School of Medicine, Tel Aviv, Israel.
REFERENCES 1. Mahadevan MM, Trounson AO, Leeton JF. Successful use
of human sperm cryobanking for in vitro fertilization. Fertil Steril 1983;40:340-3.
1256
Gamzu et aI.
Communications-in-brief
2. Yavetz H, Lessing JB, Niv Y, Amit A, Barak Y, Yovel I, et al. The efficiency of cryopreserved semen versus fresh semen for in vitro fertilization/embryo transfer. J In Vitro Fert Embryo Transf 1991;8:145-8. 3. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril1988;49:688-97. 4. Menkveld R, Stander FSH, Kotze TJW, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586-92. 5. Yavetz H, Yogev L, Homonnai Z, Paz G. Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time. Fertil Steril 1991;55:812-6. 6. Coddington CC, Franken DR, Burkman LJ, Oosthuizen WT, Kruger TF, Hodgen GD. Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl1991;12:1-8.
Fertility and Sterility
©
Vol. 58, No.6, December 1992
Printed on acid-free paper in U.S.A.
1992 The American Fertility Society
Fresh and frozen-thawed human sperm bind in a similar pattern to the zona· pellucida in the hemizona assay
Ronni Gamzu, B.Sc. * Leah Yogev, Ph.D.* Haim Yavetz, M.D.*
Zvi T. Homonnai, M.D.* Yehuda Hiss, M.D.t Gedalia Paz, Ph.D.*:j:
Institute for the Study of Fertility, Serlin Maternity Hospital, Tel Aviv Sourasky Medical Centre, Sackler Faculty of Medicine, and L. Greenberg Institute of Forensic Medicine, Tel Aviv, Israel
The successful use of frozen-thawed sperm in assisted reproductive techniques (ARTs) depends primarily on the sperm cryopreservation technique used to attain sperm cells with high fertilization capacity. Numerous investigations on cryopreserved sperm quality present contradictory conclusions. However, it is generally accepted that the freezing process impairs sperm membrane integrity and reduces the number and velocity of motile sperm cells. The in vitro fertilization (IVF) model was reported by some investigators as attaining equal fertilization rates comparing fresh with frozen-thawed sperm cells (1), whereas others described superiority of the fresh samples (2). In this study, the hemizona assay (HZA), reported as a satisfactory predictor for evaluating the fertility potential of human spermatozoa (3), was used to assess the fertility potential of fresh as opposed to frozen-thawed sperm cells of the same ejaculate. MATERIALS AND METHODS Handling of Semen
Semen was delivered from 12 different donors attending the outpatient clinic who were selected by virtue of good freezability and previous pregnancies.
Received May 11, 1992; revised and accepted July 28, 1992.
* Institute for the Study of Fertility, Serlin Maternity Hospital, Tel Aviv Sourasky Medical Centre, Sackler Faculty of Medicine. t L. Greenberg Institute of Forensic Medicine. :j: Reprint requests: Gedalia Paz, Ph.D., Institute for the Study of Fertility, Serlin Maternity Hospital, Hakirya, Post Office Box 7079, Tel Aviv, Israel 61070.
1254
Gamzu et al.
Communications-in-brief
After liquefaction, sperm concentration and motility were measured, and morphology was evaluated by strict criteria (4). An aliquot of 250 ~L of the fresh semen was twice washed with medium (Ham's F-10 medium [Flow Laboratories, Irvine, Scotland] + 1 % human serum albumin). The pellet was overlaid with 0.5 mL of medium and incubated at 37°C for 1 hour to enable swim-up separation. The remaining semen was frozen in an egg yolk (EY) containing medium, as described earlier (5). After 2 hours of freezing, one straw (0.5 mL) was thawed, and the sperm suspension was twice washed with the medium for swim-up separation as described above. After separation, both fresh and frozen-thawed samples were diluted with the medium to achieve a motile sperm concentration of 500,000 sperm cells/mL. Hemizona Assay
Oocytes obtained from the ovarian tissue of young cadavers were stored in a salt solution, following protocols described elsewhere (3). One day before the assay was carried out, the required number of oocytes was removed from the salt solution and rinsed several times in medium. Leitz micromanipulators (Leica, Wetzlar, Germany) were used for cutting the oocyte. Microblade (K-20 1730; Ktena Products, Denville, NJ) was glued to a metal bar that was bent twice at right angles to be oriented perpendicularly to the holding pipette. The cutting procedure then continued identically to that put forward by Burkman et al. (3). On the day of the assay, each half of the hemizonae pair was coincubated for 4 hours at 37°C with either fresh or frozenthawed sperm from the same sample. Hemizonae Fertility and Sterility
were then removed and rinsed with Ham's F-lO medium, and the number oftightly bound spermatozoa on the outer surface of hemizonae was counted using phase-contrast microscopy (X400). Statistical Evaluation
The sperm basic parameters are expressed as means ± SE. Statistical evaluation of the number Df tightly bound sperm in the HZA was made using the paired t-test. The hemizona index (HZ!) was calculated by the equation: (binding of fresh sperm/ binding of frozen sperm) X 100 = HZI. RESULTS
The mean concentration of the 12 original raw semen samples was 159 ± 16.9 X 106/m L. Normal forms were 19% ± 1.6%. The percent of motile sperm of the fresh and frozen-thawed samples were 46% ± 16.9% and 24% ± 3.8%, respectively. This observed superiority of the fresh samples compared with post-thaw samples, in terms of motility, was statistically significant (P < 0.005). The results ofthe HZA comparing fresh with frozen-thawed samples are given in Table 1. The number of sperm cells bound per hemizona of fresh and frozen-thawed sperm samples was similar, as indicated by the paired t-test. A distinct variation in the number of tightly bound sperm cells was observed throughout the assays, which probably derives from the differing characteristics of the zona pellucida (ZP) because of various stages of oocyte maturation. DISCUSSION
The currently widespread use of cryopreserved semen in a variety of ARTs raises the need for reassurance that the freezing-thawing process does not adversely affect the fertilizing capacity of sperm cells. However, it is commonly believed that conception rates are lower for frozen-thawed semen than for fresh semen. Also, some investigators have written about lower fertilization rates with the IVF model (2). The present study compares the binding properties of fresh versus frozen-thawed human sperm to human ZP using 12 different semen specimens from fertile men. Notwithstanding the significant reduction in motility after the freezing-thawing process, the binding capacity of the two treatments did not differ. These results are contradicted by that of Coddington et al. (6), who reported a statistically Vol. 58, No.6, December 1992
Table 1 Difference in the Binding Capacity of Fresh Versus Frozen-Thawed Sperm to the ZP in the HZA No. of sperm tightly bound to hemizonae Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 Mean ± SE
Fresh
Frozen-thawed
HZI-
48 9 14 45 82 128 86 88 88 95 150
62 8 12 38 80 118 82 96 82 88 152 66 74 ± 11.9
77.4 112.5 116.6 118.4 102.5 108.4 104.8 91.6 107.3 107.9 98.6 109.0 104.5 ± 3.2
72
75 ± 12.0
- HZI = (fresh/frozen) X 100.
insignificant reduction of approximately 30% in the binding of frozen-thawed sperm compared with the fresh samples. This discrepancy could be attributed to different freezing media and to the fact that Coddington et al. (6) used two different ejaculates as a source for the fresh and the frozen-thawed samples. It would thus seem that when frozen and thawed in EY containing media, the ability of the retrieved sperm cells to bind to the ZP is fully maintained. These conclusions are limited to high quality sperm specimens (e.g., sperm samples from donors); the response of low quality sperm specimens to this process has yet to be evaluated. Our results also support the possible use of frozenthawed donor human sperm specimens as controls in the HZA procedure. This should enable an assay of greater control that could be put to use as a routine diagnostic tool. SUMMARY
This study evaluated the impact of sperm cryopreservation on sperm quality. The HZA was used to test the binding capacity of fresh as opposed to frozen-thawed sperm from 12 donors. Fresh and frozen-thawed sperm motility was 47% ± 1.5% and 24% ± 3.8% (mean ± SE), respectively. However, the number of sperm cells attached to the hemizonae was 75 ± 12.0 and 74 ± 11.9, respectively. We conclude that cryopreservation results in a reduced number of motile sperm cells but does not adversely affect the ability of rescued sperm cells to bind to Gamzu et al.
Communications-in-brief
1255
the ZP. The study also supports the use of frozenthawed rather than fresh donor sperm for control in the HZA procedure. Key Words: Cryopreservation, zona pellucida, hemizona assay. Acknowledgment. We greatly appreciate the scientific and editorial advising of Dov Lichtenberg, Ph.D., Physiology and Pharmacology Department, Sackler School of Medicine, Tel Aviv, Israel.
REFERENCES 1. Mahadevan MM, Trounson AO, Leeton JF. Successful use
of human sperm cryobanking for in vitro fertilization. Fertil Steril 1983;40:340-3.
1256
Gamzu et aI.
Communications-in-brief
2. Yavetz H, Lessing JB, Niv Y, Amit A, Barak Y, Yovel I, et al. The efficiency of cryopreserved semen versus fresh semen for in vitro fertilization/embryo transfer. J In Vitro Fert Embryo Transf 1991;8:145-8. 3. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril1988;49:688-97. 4. Menkveld R, Stander FSH, Kotze TJW, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586-92. 5. Yavetz H, Yogev L, Homonnai Z, Paz G. Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time. Fertil Steril 1991;55:812-6. 6. Coddington CC, Franken DR, Burkman LJ, Oosthuizen WT, Kruger TF, Hodgen GD. Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl1991;12:1-8.
Fertility and Sterility
