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Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae
FERTILITY AND STERILITY威 VOL. 73, NO. 4, APRIL 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae Yao-Yuan Hsieh, M.D., Horng-Der Tsai, M.D., Chi-Chen Chang, M.D., and Hui-Yu Lo, M.D. Department of Obstetrics and Gynecology, China Medical College Hospital, Taichung, Taiwan
Objective: To compare the empty zona pellucidae (ZP) of different species for use in the cryopreservation of spermatozoa. Design: Prospective study. Setting: An IVF unit of a medical center. Patient(s): Patients with azoospermia, oligoasthenozoospermia, or normal spermatozoa. Intervention(s): Human and mouse ZP were prepared by evacuating the cytoplasm of oocytes or embryos. The evacuated ZP were injected with spermatozoa from patients with severe oligoasthenozoospermia and from healthy, fertile men. After the freezing and thawing procedure, the spermatozoa were aspirated outside the ZP. Main Outcome Measure(s): The number of spermatozoa per ZP, the number of motile sperm before freezing and after thawing, the number of sperm lost per ZP after freezing, and the sperm recovery rate were compared according to the different origins of the ZP and the sperm. Result(s): The number of spermatozoa, number of motile sperm before freezing and after thawing, number of sperm lost per ZP, and sperm recovery rate were comparable in all groups. The total mean number of motile sperm before freezing and after thawing, the mean number of nonmotile sperm after thawing, the mean number of sperm lost after thawing, and the sperm recovery rate were 14.5%, 11.8%, 1.0%, 1.5%, and 82%, respectively. Conclusion(s): Zona pellucidae are an ideal vehicle for the cryopreservation of sperm collected by testicular sperm extraction or microsurgical epididymal sperm aspiration or from patients with severe oligoasthenozoospermia. There were no differences when human and mouse ZP were used for sperm storage. (Fertil Steril威 2000;73:694 – 8. ©2000 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, empty zona pellucidae, oligoasthenozoospermia, spermatozoa, zona pellucidae
Received August 10, 1999; revised and accepted October 21, 1999. Supported by China Medical College Hospital, Taichung, Taiwan. Reprint requests: HorngDer Tsai, M.D., Department of Obstetrics and Gynecology, China Medical College Hospital, No. 2 Yuh-Der Road, Taichung, Taiwan (FAX: 886-42023257; E-mail: d3531 @hpd.cmch.org.tw). 0015-0282/00/$20.00 PII S0015-0282(99)00612-3
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Men who have azoospermia can now be treated through the surgical isolation of spermatozoa using microsurgical epididymal sperm aspiration (MESA) or testicular sperm extraction (TESE) (1). However, multiple testicular operations are not only costly but may lead to adverse physiologic effects and possible testicular failure (2). The need to repeat these procedures can be avoided by the use of sperm cryopreservation (3). However, conventional sperm suspension cryopreservation is not suitable for patients with severe oligoasthenospermia or azoospermia. After the traditional freezing and thawing procedure, most spermatozoa are diluted and lost during centrifugation, making identification difficult. The cryopreservation of non-
dissected seminiferous tubules still requires lengthy and uncertain sample preparation on the day of retrieval. Cohen et al. (4) first demonstrated that the zona pellucida (ZP) is an ideal vehicle for sperm cryopreservation. After removal of the cellular material from oocytes or embryos, the empty ZP provides a suitable vehicle for preserving the few spermatozoa that can be obtained from patients with severe male infertility. In this series, we modified the method of Cohen et al. (4), making it more effective and simpler. We then compared the evacuated oocytes or embryos of humans and mice for use in the cryopreservation of spermatozoa. We also compared the influence of cryopreserva-
tion in ZP on sperm obtained by TESE or MESA and sperm obtained from patients with oligoasthenospermia and healthy, fertile men.
MATERIALS AND METHODS Between October 1998 and March 1999, all patients who presented to our center with severe male infertility, including azoospermia, oligozoospermia (⬍1 ⫻ 106/mL), and asthenozoospermia (⬍30% motile sperm), were enrolled in this study. Semen was obtained from healthy, fertile men for the control analysis. Zona pellucidae were obtained from human oocytes, including immature (germinal vesicle, metaphase I stage), unfertilized, and abnormally fertilized (three pronuclei) oocytes; human embryos (two- to eight-cell stage), including developing-arrested embryos during prolonged culture and donated cryopreserved embryos; mouse oocytes; and mouse embryos. All donated human oocytes and embryos were inseminated using the intracytoplasmic sperm injection procedure. The study was approved by the ethics committee of the China Medical College Hospital. Approval had been obtained from the Institutional Review Board before this series of patients was enrolled. Written informed consent was provided by all the couples who donated their cryopreserved embryos, abnormally developing oocytes, and sperm. Oocytes or embryos were obtained after superovulation from 10- to 12-week-old CB6F1 hybrid female mice, either mated or nonmated (oocytes were obtained from nonmated mice), by excision and flushing of their oviducts. According to the different origins of the ZP and sperm, 12 groups were created: [1] human oocyte and normal sperm; [2] human oocyte and sperm obtained from a patient with oligoasthenospermia; [3] human oocyte and sperm obtained by MESA or TESE; [4] human embryo and normal sperm; [5] human embryo and sperm obtained from a patient with oligoasthenospermia; [6] human embryo and sperm obtained by MESA or TESE; [7] mouse oocyte and normal sperm; [8] mouse oocyte and sperm obtained from a patient with oligoasthenospermia; [9] mouse oocyte and sperm obtained by MESA or TESE; [10] mouse embryo and normal sperm; [11] mouse embryo and sperm obtained from a patient with oligoasthenospermia; and [12] mouse embryo and sperm obtained by MESA or TESE. The different types of human oocytes (immature, unfertilized, and abnormally fertilized) and embryos (arrested development or donated cryopreserved) were divided evenly for the preservation of the different types of semen (normal semen, semen with severe oligoasthenospermia, and semen with azoospermia). The sperm obtained from patients with obstructed and nonobstructed azoospermia were allocated evenly to each group. All micromanipulations were performed under HEPESFERTILITY & STERILITY威
buffered human tubal fluid (HEPES-HTF). Micromanipulations were performed in shallow Falcon 1006 dishes (Becton Dickinson Labware, Franklin Lakes, NJ) using 5-L droplets of HEPES-HTF according to the method of Palermo et al. (5). The holding (60 – 80 m) and injection (4 – 8 m) micropipettes used for the holding and evacuation of oocytes or embryos, aspiration of sperm, and injection of sperm were commercial products (Humagen, Charlottesville, VA). The micromanipulations were performed on a Nikon inverted microscope (Nikon, Tokyo, Japan) with a micropipette connected to Narishige micromanipulators (MM188; Tokyo, Japan). While holding the oocytes or embryos, the injection pipette was moved through the oolemma and the cytoplasm was aspirated until the ZP was empty. At times, the injection pipette had to be changed or withdrawn from the ZP to remove the sticky cytoplasm from the tip of the pipette. After aspiration of the cytoplasm, the empty ZP frequently collapsed. The collapsed ZP was transferred to a 10% glycerol solution and reinflation was noted later. The sperm obtained by TESE were placed into HEPESHTF medium and dissected using two glass slides to release the contents of the seminiferous tubules. After thorough dissection and the addition of 5 mL of HEPES-HTF, the specimen was centrifuged for 5 minutes at 1,800 ⫻ g. The semen obtained by MESA or from fresh ejaculates was processed by centrifugation at 1,800 ⫻ g and the mini-Percoll technique as described by Palermo et al. (6). The spermatozoa from healthy, fertile men were prepared by the Percoll method using three different density gradients (95%, 70%, and 50%). After centrifugation, 1 L of the sperm suspension was diluted with HEPES-HTF until the sperm concentration was approximately 100 –1,000 per milliliter. When the sperm concentration and/or motility was adequate, the sperm were obtained by aspiration through the injection pipette. After fixation of the ZP by a holding pipette, the spermatozoa were aspirated with the injection pipette and injected into the ZP (Fig. 1). The sperm were released slowly from the injection pipette to minimize the inflation of the ZP. After injection of the sperm, the ZP was observed closely to prevent the sperm from escaping. Then the individual motile spermatozoa were counted three times before being cryopreserved (Fig. 2). The injected ZP was placed in a 60% glycerol solution. Before ZP loading, a 0.25-mL, clear plastic insemination straw (I.M.V., l’Aigle, France) was marked with the patient’s name and date, and rinsed three times with glycerol solution. The straw was loaded with 40 L of glycerol in the middle part and separated from 20 L of glycerol on both ends with air bubbles. The ZP was loaded to the middle part of the glycerol and heat-sealed at both ends. Then the straw was exposed to liquid nitrogen vapor for 30 minutes and plunged into liquid nitrogen. The straws were kept in liquid nitrogen for ⱖ24 hours. 695
FIGURE 1 Injection of spermatozoa with an intracytoplasmic sperm injection needle into an empty ZP of a prefertilization human oocyte.
(range, 9.3–14.4), respectively. The mean number of nonmotile sperm after thawing was 1.0 (range, 0.8 –1.6). The mean number of lost or trapped spermatozoa per ZP was 1.5 (range, 1.3–2.1). The sperm recovery rate was 82% (range, 76%– 87%). The mean number of motile sperm before freezing and after thawing, the mean number of nonmotile sperm after thawing, the number of sperm lost after thawing, and the sperm recovery rate in each group were statistically comparable.
DISCUSSION
Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
Subsequently, the straws were thawed by immersion in a 37°C water bath for 6 seconds. One end of the straw was cut and a 16-gauge needle of a syringe was inserted. The other end then was cut and the medium was expelled into HEPESHTF medium. The ZP was transferred into an intracytoplasmic sperm injection dish containing droplets of HEPESHTF. The spermatozoa inside the ZP then were aspirated with the injection pipette. The numbers of motile, nonmotile, lost, and trapped spermatozoa per ZP were recorded. The rate of recovery of motile spermatozoa was calculated. The number of spermatozoa per ZP, the number of motile sperm before freezing and after thawing, the number of sperm lost per ZP after freezing, and the rate of recovery of motile sperm in each group were compared. Data comparisons were made using 2 analysis.
The collection of sperm from TESE specimens is timeconsuming. Poor MESA specimens or ejaculates from men with severe oligoasthenospermia also require lengthy sperm preparation. Further, repeated TESE or MESA procedures may have adverse effects on the testis, including deterioration of spermatogenesis, inflammation, irreversible atrophy, and partial testicular devascularization (7). Conventional sperm freezing is not suitable for specimens with a limited number of sperm (8, 9). The cryopreservation of MESA and TESE specimens is time-consuming and the availability of sperm is uncertain (8, 9). Cryopreservation in ZP avoids the loss of sperm that occurs with the dilution and washing of sperm during conventional cryopreservation procedures (10). The rate of recovery of motile sperm after cryopreservation in ZP is higher than after conventional cryopreservation with cryoseeds and dithiothreitol (10 –12). With cryopreservation in ZP, TESE or MESA could be performed without regard to the timing of egg retrieval. It has been demonstrated that the preservation of numer-
FIGURE 2 Aggregation of spermatozoa within the empty ZP after sperm injection.
RESULTS A total of 172 empty ZP were used for these experiments, including 33 from human oocytes, 30 from human embryos, 75 from mouse oocytes, and 34 from mouse embryos. The sperm obtained by TESE and MESA were retrieved from 5 patients, including 3 patients with obstructive azoospermia and 2 patients with nonobstructive azoospermia. The fresh ejaculates were obtained from 11 patients with severe oligoasthenozoospermia and 3 healthy, fertile men. A total of 2,524 motile spermatozoa were injected into empty ZP (Table 1). The mean numbers of motile sperm per ZP before freezing and after thawing were 14.5 (range, 12.1–17.2) and 11.8 696
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Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
Vol. 73, No. 4, April 2000
TABLE 1 Number of motile spermatozoa before and after freezing, number of sperm lost after freezing, and rate of sperm recovery in human and mouse ZP. Total no. of motile sperm (mean no. of motile sperm per ZP) Source of ZP Human oocyte
Human embryo
Mouse oocyte
Mouse embryo
Origin of sperm Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or
TESE
TESE
TESE
TESE
Total
ZP no.
Before freezing
After thawing
Mean no. of nonmotile sperm after thawing (no. of nonmotile sperm/no. of ZP)
13 15 5 14 12 4 31 36 8 15 13 6 172
202 (15.5) 254 (16.9) 62 (12.4) 241 (17.2) 193 (16.1) 53 (13.3) 435 (14.0) 496 (13.8) 97 (12.1) 221 (14.7) 196 (15.1) 74 (12.3) 210.3 (14.5)
171 (13.2) 208 (13.9) 48 (9.6) 202 (14.4) 155 (12.9) 42 (10.5) 379 (12.2) 405 (11.3) 74 (9.3) 179 (11.9) 162 (12.5) 61 (10.2) 173.8 (11.8)
1.2 (15/13) 1.6 (24/15) 1.2 (6/5) 1.0 (14/14) 1.1 (13/12) 1.3 (5/4) 0.4 (12/31) 1.0 (35/36) 1.1 (9/8) 1.1 (16/15) 0.8 (10/13) 0.8 (5/6) 1.0 (164/172)
Mean no. of sperm lost after thawing (no. of sperm/ no. of ZP)
Sperm recovery rate (%)
1.2 (16/13) 1.5 (22/15) 1.6 (8/5) 1.8 (25/14) 2.1 (25/12) 1.5 (6/4) 1.4 (44/31) 1.6 (56/36) 1.8 (14/8) 1.7 (26/15) 1.9 (24/13) 1.3 (8/6) 1.5 (266/172)
85 82 77 84 80 79 87 82 76 81 83 82 82
Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
ous sperm may have a deleterious effect on sperm during freezing, thawing, and centrifugation (13, 14). Cohen et al. (4) demonstrated that 1–3 spermatozoa per ZP is optimal. In this series, we preserved 15–20 sperm per ZP. Our sperm recovery rates were comparable to those obtained by Cohen et al. (4) with single sperm cryopreservation. With our method, only one or two ZP are needed to provide adequate sperm for intracytoplasmic sperm injection in most cases. Although the aggregation of spermatozoa can inhibit individual motility, the sperm became more motile after aspiration and exposure to medium. If the ZP is not evacuated completely, the number of trapped sperm increases and the sperm recovery rate decreases. Our findings disagree with the findings of Cohen et al. (4), who reported a higher sperm recovery rate and lower number of trapped sperm in the mouse ZP than in the human ZP. After complete evacuation of the cytoplasm, we observed similar rates of recovery of motile sperm from human and mouse ZP. The disadvantages of the ZP of the CB6F1 mouse for sperm preservation are its small size and translucency, which may result in a higher rate of loss of whole ZP during the cryopreservation procedure. With adequate training, such loss could be decreased. However, the mouse ZP has the advantage of better availability. The sperm collected by TESE or MESA had slightly lower motility after cryopreservation in ZP than the sperm obtained from men with oligoasthenozoospermia or normal sperm quality. However, their postthaw motility levels were not statistically different. After evacuation of the cytoplasm, we immersed the collapsed ZP in 10% glycerol for inflation FERTILITY & STERILITY威
instead of injecting it with medium. This method is easy as well as time- and labor-saving. We did not use polyvinylpyrrolidone for the retrieval of spermatozoa. The motility of sperm obtained by MESA or TESE, or from patients with oligoasthenospermia is poor. Cohen et al. (4) reported that the number of spermatozoa lost through holes after thawing ranged from 10%–37%. In this series, most sperm loss was due to trapping by the residual cytoplasm in the ZP. This difference is related to our use of 4- to 8-m pipettes instead of the 15-m micropipettes or Tyrode’s solution used by Cohen et al. (4) for evacuation of the cytoplasm. The 4- to 8-m pipettes have the advantage of avoiding the escape of sperm through the holes made by the 15-m pipettes. We experienced insignificant difficulty in evacuating the cytoplasm with 4- to 8-m pipettes. In conclusion, an evacuated ZP is an ideal vehicle for sperm storage. This series provides a database for comparing sperm and ZP of different origins for use in cryopreservation. Our modified method for cryopreservation in ZP is both easier and quicker. However, a larger study is necessary to confirm these findings. References 1. Schoysman R, Vanderzwalmen P, Segal-Bertin G, van de Casseye M. Successful fertilization by testicular spermatozoa in an in-vitro fertilization programme. Hum Reprod 1993;8:1339 – 40. 2. Schlegel PR, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997;12:1688 –92. 3. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa from testicular biopsy. Hum Reprod 1995; 10:148 –52. 4. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human spermatozoa. Hum Reprod 1997;12:994 –1001.
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5. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic sperm injection of single spermatozoa into an oocyte. Lancet 1992;340:17– 8. 6. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995;63:1231– 40. 7. Schlegel P. Physiologic consequences of TESE [abstract]. Hum Reprod 1996;11:159. 8. Patrizio P, Ord T, Balmaceda JP, Asch RH. Successful fertilization, pregnancy, and birth using epididymal sperm frozen 24 hours after conventional oocyte insemination. Fertil Steril 1995;64:863–5. 9. Podsiadly BT, Woolcott RJ, Stanger JD, Stevenson K. Case report pregnancy resulting from intracytoplasmic sperm injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum Reprod 1996;11:1306 – 8.
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10. Verheyen G, Pletincx I, Van Steirteghem A. Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high quality human sperm. Hum Reprod 1993;8:1678 – 84. 11. Bongso A, Jarina AK, Ho J, Ng SC, Ratnam SS. Comparative evaluation of three sperm-washing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples. Arch Androl 1993;31:223–30. 12. Sawetawan C, Bruns ES, Gail SP. Improvement of post-thaw motility in poor quality human semen. Fertil Steril 1993;60:706 –10. 13. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988;9:367–76. 14. Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore 1992;21:517–24.
Vol. 73, No. 4, April 2000
Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae Yao-Yuan Hsieh, M.D., Horng-Der Tsai, M.D., Chi-Chen Chang, M.D., and Hui-Yu Lo, M.D. Department of Obstetrics and Gynecology, China Medical College Hospital, Taichung, Taiwan
Objective: To compare the empty zona pellucidae (ZP) of different species for use in the cryopreservation of spermatozoa. Design: Prospective study. Setting: An IVF unit of a medical center. Patient(s): Patients with azoospermia, oligoasthenozoospermia, or normal spermatozoa. Intervention(s): Human and mouse ZP were prepared by evacuating the cytoplasm of oocytes or embryos. The evacuated ZP were injected with spermatozoa from patients with severe oligoasthenozoospermia and from healthy, fertile men. After the freezing and thawing procedure, the spermatozoa were aspirated outside the ZP. Main Outcome Measure(s): The number of spermatozoa per ZP, the number of motile sperm before freezing and after thawing, the number of sperm lost per ZP after freezing, and the sperm recovery rate were compared according to the different origins of the ZP and the sperm. Result(s): The number of spermatozoa, number of motile sperm before freezing and after thawing, number of sperm lost per ZP, and sperm recovery rate were comparable in all groups. The total mean number of motile sperm before freezing and after thawing, the mean number of nonmotile sperm after thawing, the mean number of sperm lost after thawing, and the sperm recovery rate were 14.5%, 11.8%, 1.0%, 1.5%, and 82%, respectively. Conclusion(s): Zona pellucidae are an ideal vehicle for the cryopreservation of sperm collected by testicular sperm extraction or microsurgical epididymal sperm aspiration or from patients with severe oligoasthenozoospermia. There were no differences when human and mouse ZP were used for sperm storage. (Fertil Steril威 2000;73:694 – 8. ©2000 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, empty zona pellucidae, oligoasthenozoospermia, spermatozoa, zona pellucidae
Received August 10, 1999; revised and accepted October 21, 1999. Supported by China Medical College Hospital, Taichung, Taiwan. Reprint requests: HorngDer Tsai, M.D., Department of Obstetrics and Gynecology, China Medical College Hospital, No. 2 Yuh-Der Road, Taichung, Taiwan (FAX: 886-42023257; E-mail: d3531 @hpd.cmch.org.tw). 0015-0282/00/$20.00 PII S0015-0282(99)00612-3
694
Men who have azoospermia can now be treated through the surgical isolation of spermatozoa using microsurgical epididymal sperm aspiration (MESA) or testicular sperm extraction (TESE) (1). However, multiple testicular operations are not only costly but may lead to adverse physiologic effects and possible testicular failure (2). The need to repeat these procedures can be avoided by the use of sperm cryopreservation (3). However, conventional sperm suspension cryopreservation is not suitable for patients with severe oligoasthenospermia or azoospermia. After the traditional freezing and thawing procedure, most spermatozoa are diluted and lost during centrifugation, making identification difficult. The cryopreservation of non-
dissected seminiferous tubules still requires lengthy and uncertain sample preparation on the day of retrieval. Cohen et al. (4) first demonstrated that the zona pellucida (ZP) is an ideal vehicle for sperm cryopreservation. After removal of the cellular material from oocytes or embryos, the empty ZP provides a suitable vehicle for preserving the few spermatozoa that can be obtained from patients with severe male infertility. In this series, we modified the method of Cohen et al. (4), making it more effective and simpler. We then compared the evacuated oocytes or embryos of humans and mice for use in the cryopreservation of spermatozoa. We also compared the influence of cryopreserva-
tion in ZP on sperm obtained by TESE or MESA and sperm obtained from patients with oligoasthenospermia and healthy, fertile men.
MATERIALS AND METHODS Between October 1998 and March 1999, all patients who presented to our center with severe male infertility, including azoospermia, oligozoospermia (⬍1 ⫻ 106/mL), and asthenozoospermia (⬍30% motile sperm), were enrolled in this study. Semen was obtained from healthy, fertile men for the control analysis. Zona pellucidae were obtained from human oocytes, including immature (germinal vesicle, metaphase I stage), unfertilized, and abnormally fertilized (three pronuclei) oocytes; human embryos (two- to eight-cell stage), including developing-arrested embryos during prolonged culture and donated cryopreserved embryos; mouse oocytes; and mouse embryos. All donated human oocytes and embryos were inseminated using the intracytoplasmic sperm injection procedure. The study was approved by the ethics committee of the China Medical College Hospital. Approval had been obtained from the Institutional Review Board before this series of patients was enrolled. Written informed consent was provided by all the couples who donated their cryopreserved embryos, abnormally developing oocytes, and sperm. Oocytes or embryos were obtained after superovulation from 10- to 12-week-old CB6F1 hybrid female mice, either mated or nonmated (oocytes were obtained from nonmated mice), by excision and flushing of their oviducts. According to the different origins of the ZP and sperm, 12 groups were created: [1] human oocyte and normal sperm; [2] human oocyte and sperm obtained from a patient with oligoasthenospermia; [3] human oocyte and sperm obtained by MESA or TESE; [4] human embryo and normal sperm; [5] human embryo and sperm obtained from a patient with oligoasthenospermia; [6] human embryo and sperm obtained by MESA or TESE; [7] mouse oocyte and normal sperm; [8] mouse oocyte and sperm obtained from a patient with oligoasthenospermia; [9] mouse oocyte and sperm obtained by MESA or TESE; [10] mouse embryo and normal sperm; [11] mouse embryo and sperm obtained from a patient with oligoasthenospermia; and [12] mouse embryo and sperm obtained by MESA or TESE. The different types of human oocytes (immature, unfertilized, and abnormally fertilized) and embryos (arrested development or donated cryopreserved) were divided evenly for the preservation of the different types of semen (normal semen, semen with severe oligoasthenospermia, and semen with azoospermia). The sperm obtained from patients with obstructed and nonobstructed azoospermia were allocated evenly to each group. All micromanipulations were performed under HEPESFERTILITY & STERILITY威
buffered human tubal fluid (HEPES-HTF). Micromanipulations were performed in shallow Falcon 1006 dishes (Becton Dickinson Labware, Franklin Lakes, NJ) using 5-L droplets of HEPES-HTF according to the method of Palermo et al. (5). The holding (60 – 80 m) and injection (4 – 8 m) micropipettes used for the holding and evacuation of oocytes or embryos, aspiration of sperm, and injection of sperm were commercial products (Humagen, Charlottesville, VA). The micromanipulations were performed on a Nikon inverted microscope (Nikon, Tokyo, Japan) with a micropipette connected to Narishige micromanipulators (MM188; Tokyo, Japan). While holding the oocytes or embryos, the injection pipette was moved through the oolemma and the cytoplasm was aspirated until the ZP was empty. At times, the injection pipette had to be changed or withdrawn from the ZP to remove the sticky cytoplasm from the tip of the pipette. After aspiration of the cytoplasm, the empty ZP frequently collapsed. The collapsed ZP was transferred to a 10% glycerol solution and reinflation was noted later. The sperm obtained by TESE were placed into HEPESHTF medium and dissected using two glass slides to release the contents of the seminiferous tubules. After thorough dissection and the addition of 5 mL of HEPES-HTF, the specimen was centrifuged for 5 minutes at 1,800 ⫻ g. The semen obtained by MESA or from fresh ejaculates was processed by centrifugation at 1,800 ⫻ g and the mini-Percoll technique as described by Palermo et al. (6). The spermatozoa from healthy, fertile men were prepared by the Percoll method using three different density gradients (95%, 70%, and 50%). After centrifugation, 1 L of the sperm suspension was diluted with HEPES-HTF until the sperm concentration was approximately 100 –1,000 per milliliter. When the sperm concentration and/or motility was adequate, the sperm were obtained by aspiration through the injection pipette. After fixation of the ZP by a holding pipette, the spermatozoa were aspirated with the injection pipette and injected into the ZP (Fig. 1). The sperm were released slowly from the injection pipette to minimize the inflation of the ZP. After injection of the sperm, the ZP was observed closely to prevent the sperm from escaping. Then the individual motile spermatozoa were counted three times before being cryopreserved (Fig. 2). The injected ZP was placed in a 60% glycerol solution. Before ZP loading, a 0.25-mL, clear plastic insemination straw (I.M.V., l’Aigle, France) was marked with the patient’s name and date, and rinsed three times with glycerol solution. The straw was loaded with 40 L of glycerol in the middle part and separated from 20 L of glycerol on both ends with air bubbles. The ZP was loaded to the middle part of the glycerol and heat-sealed at both ends. Then the straw was exposed to liquid nitrogen vapor for 30 minutes and plunged into liquid nitrogen. The straws were kept in liquid nitrogen for ⱖ24 hours. 695
FIGURE 1 Injection of spermatozoa with an intracytoplasmic sperm injection needle into an empty ZP of a prefertilization human oocyte.
(range, 9.3–14.4), respectively. The mean number of nonmotile sperm after thawing was 1.0 (range, 0.8 –1.6). The mean number of lost or trapped spermatozoa per ZP was 1.5 (range, 1.3–2.1). The sperm recovery rate was 82% (range, 76%– 87%). The mean number of motile sperm before freezing and after thawing, the mean number of nonmotile sperm after thawing, the number of sperm lost after thawing, and the sperm recovery rate in each group were statistically comparable.
DISCUSSION
Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
Subsequently, the straws were thawed by immersion in a 37°C water bath for 6 seconds. One end of the straw was cut and a 16-gauge needle of a syringe was inserted. The other end then was cut and the medium was expelled into HEPESHTF medium. The ZP was transferred into an intracytoplasmic sperm injection dish containing droplets of HEPESHTF. The spermatozoa inside the ZP then were aspirated with the injection pipette. The numbers of motile, nonmotile, lost, and trapped spermatozoa per ZP were recorded. The rate of recovery of motile spermatozoa was calculated. The number of spermatozoa per ZP, the number of motile sperm before freezing and after thawing, the number of sperm lost per ZP after freezing, and the rate of recovery of motile sperm in each group were compared. Data comparisons were made using 2 analysis.
The collection of sperm from TESE specimens is timeconsuming. Poor MESA specimens or ejaculates from men with severe oligoasthenospermia also require lengthy sperm preparation. Further, repeated TESE or MESA procedures may have adverse effects on the testis, including deterioration of spermatogenesis, inflammation, irreversible atrophy, and partial testicular devascularization (7). Conventional sperm freezing is not suitable for specimens with a limited number of sperm (8, 9). The cryopreservation of MESA and TESE specimens is time-consuming and the availability of sperm is uncertain (8, 9). Cryopreservation in ZP avoids the loss of sperm that occurs with the dilution and washing of sperm during conventional cryopreservation procedures (10). The rate of recovery of motile sperm after cryopreservation in ZP is higher than after conventional cryopreservation with cryoseeds and dithiothreitol (10 –12). With cryopreservation in ZP, TESE or MESA could be performed without regard to the timing of egg retrieval. It has been demonstrated that the preservation of numer-
FIGURE 2 Aggregation of spermatozoa within the empty ZP after sperm injection.
RESULTS A total of 172 empty ZP were used for these experiments, including 33 from human oocytes, 30 from human embryos, 75 from mouse oocytes, and 34 from mouse embryos. The sperm obtained by TESE and MESA were retrieved from 5 patients, including 3 patients with obstructive azoospermia and 2 patients with nonobstructive azoospermia. The fresh ejaculates were obtained from 11 patients with severe oligoasthenozoospermia and 3 healthy, fertile men. A total of 2,524 motile spermatozoa were injected into empty ZP (Table 1). The mean numbers of motile sperm per ZP before freezing and after thawing were 14.5 (range, 12.1–17.2) and 11.8 696
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Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
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TABLE 1 Number of motile spermatozoa before and after freezing, number of sperm lost after freezing, and rate of sperm recovery in human and mouse ZP. Total no. of motile sperm (mean no. of motile sperm per ZP) Source of ZP Human oocyte
Human embryo
Mouse oocyte
Mouse embryo
Origin of sperm Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or Normal semen Severe oligoasthenospermia Azoospermia after MESA or
TESE
TESE
TESE
TESE
Total
ZP no.
Before freezing
After thawing
Mean no. of nonmotile sperm after thawing (no. of nonmotile sperm/no. of ZP)
13 15 5 14 12 4 31 36 8 15 13 6 172
202 (15.5) 254 (16.9) 62 (12.4) 241 (17.2) 193 (16.1) 53 (13.3) 435 (14.0) 496 (13.8) 97 (12.1) 221 (14.7) 196 (15.1) 74 (12.3) 210.3 (14.5)
171 (13.2) 208 (13.9) 48 (9.6) 202 (14.4) 155 (12.9) 42 (10.5) 379 (12.2) 405 (11.3) 74 (9.3) 179 (11.9) 162 (12.5) 61 (10.2) 173.8 (11.8)
1.2 (15/13) 1.6 (24/15) 1.2 (6/5) 1.0 (14/14) 1.1 (13/12) 1.3 (5/4) 0.4 (12/31) 1.0 (35/36) 1.1 (9/8) 1.1 (16/15) 0.8 (10/13) 0.8 (5/6) 1.0 (164/172)
Mean no. of sperm lost after thawing (no. of sperm/ no. of ZP)
Sperm recovery rate (%)
1.2 (16/13) 1.5 (22/15) 1.6 (8/5) 1.8 (25/14) 2.1 (25/12) 1.5 (6/4) 1.4 (44/31) 1.6 (56/36) 1.8 (14/8) 1.7 (26/15) 1.9 (24/13) 1.3 (8/6) 1.5 (266/172)
85 82 77 84 80 79 87 82 76 81 83 82 82
Hsieh. Cryopreservation human spermatozoa. Fertil Steril 2000.
ous sperm may have a deleterious effect on sperm during freezing, thawing, and centrifugation (13, 14). Cohen et al. (4) demonstrated that 1–3 spermatozoa per ZP is optimal. In this series, we preserved 15–20 sperm per ZP. Our sperm recovery rates were comparable to those obtained by Cohen et al. (4) with single sperm cryopreservation. With our method, only one or two ZP are needed to provide adequate sperm for intracytoplasmic sperm injection in most cases. Although the aggregation of spermatozoa can inhibit individual motility, the sperm became more motile after aspiration and exposure to medium. If the ZP is not evacuated completely, the number of trapped sperm increases and the sperm recovery rate decreases. Our findings disagree with the findings of Cohen et al. (4), who reported a higher sperm recovery rate and lower number of trapped sperm in the mouse ZP than in the human ZP. After complete evacuation of the cytoplasm, we observed similar rates of recovery of motile sperm from human and mouse ZP. The disadvantages of the ZP of the CB6F1 mouse for sperm preservation are its small size and translucency, which may result in a higher rate of loss of whole ZP during the cryopreservation procedure. With adequate training, such loss could be decreased. However, the mouse ZP has the advantage of better availability. The sperm collected by TESE or MESA had slightly lower motility after cryopreservation in ZP than the sperm obtained from men with oligoasthenozoospermia or normal sperm quality. However, their postthaw motility levels were not statistically different. After evacuation of the cytoplasm, we immersed the collapsed ZP in 10% glycerol for inflation FERTILITY & STERILITY威
instead of injecting it with medium. This method is easy as well as time- and labor-saving. We did not use polyvinylpyrrolidone for the retrieval of spermatozoa. The motility of sperm obtained by MESA or TESE, or from patients with oligoasthenospermia is poor. Cohen et al. (4) reported that the number of spermatozoa lost through holes after thawing ranged from 10%–37%. In this series, most sperm loss was due to trapping by the residual cytoplasm in the ZP. This difference is related to our use of 4- to 8-m pipettes instead of the 15-m micropipettes or Tyrode’s solution used by Cohen et al. (4) for evacuation of the cytoplasm. The 4- to 8-m pipettes have the advantage of avoiding the escape of sperm through the holes made by the 15-m pipettes. We experienced insignificant difficulty in evacuating the cytoplasm with 4- to 8-m pipettes. In conclusion, an evacuated ZP is an ideal vehicle for sperm storage. This series provides a database for comparing sperm and ZP of different origins for use in cryopreservation. Our modified method for cryopreservation in ZP is both easier and quicker. However, a larger study is necessary to confirm these findings. References 1. Schoysman R, Vanderzwalmen P, Segal-Bertin G, van de Casseye M. Successful fertilization by testicular spermatozoa in an in-vitro fertilization programme. Hum Reprod 1993;8:1339 – 40. 2. Schlegel PR, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997;12:1688 –92. 3. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa from testicular biopsy. Hum Reprod 1995; 10:148 –52. 4. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human spermatozoa. Hum Reprod 1997;12:994 –1001.
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5. Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmic sperm injection of single spermatozoa into an oocyte. Lancet 1992;340:17– 8. 6. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995;63:1231– 40. 7. Schlegel P. Physiologic consequences of TESE [abstract]. Hum Reprod 1996;11:159. 8. Patrizio P, Ord T, Balmaceda JP, Asch RH. Successful fertilization, pregnancy, and birth using epididymal sperm frozen 24 hours after conventional oocyte insemination. Fertil Steril 1995;64:863–5. 9. Podsiadly BT, Woolcott RJ, Stanger JD, Stevenson K. Case report pregnancy resulting from intracytoplasmic sperm injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum Reprod 1996;11:1306 – 8.
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10. Verheyen G, Pletincx I, Van Steirteghem A. Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high quality human sperm. Hum Reprod 1993;8:1678 – 84. 11. Bongso A, Jarina AK, Ho J, Ng SC, Ratnam SS. Comparative evaluation of three sperm-washing methods to improve sperm concentration and motility in frozen-thawed oligozoospermic and normozoospermic samples. Arch Androl 1993;31:223–30. 12. Sawetawan C, Bruns ES, Gail SP. Improvement of post-thaw motility in poor quality human semen. Fertil Steril 1993;60:706 –10. 13. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988;9:367–76. 14. Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore 1992;21:517–24.
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