1 Department of Gynecology, Institute of Obstetrics and Gynecology, 2 CryobankInfertility Center, Bialystok, Poland, 3 Infertility Center, Department of Obstetrics and Gynecology, Ghent University Hospital, Belgium and 4 Department of Gynecological Endocrinology, Institute of Obstetrics and Gynecology, Bialystok, Poland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: sperm cryopreservation/ICSI/fertilization/pregnancy/prospective randomized study
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
With the advent of intracytoplasmic sperm injection (ICSI) only a small number of motile spermatozoa are required for a successful fertilization (Palermo et al., 1992). Indeed, ICSI can be successfully performed with fresh and frozenthawed spermatozoa from ejaculated semen from patients with oligoasthenoteratozoospermia (OAT) or from spermatozoa extracted from the epididymis or testis in cases of obstructive or non-obstructive azoospermia (Ng et al., 1991
, Tournaye et al., 1994
; Devroey et al., 1996
; Friedler et al., 1997
). Although there is no doubt that frozenthawed spermatozoa can be used to fertilize oocytes by ICSI, a prospective randomized study comparing the efficacy of ICSI with either fresh or frozenthawed ejaculated spermatozoa from patients with OAT has not yet been performed. Data from such a study are needed because it is known that the freezingthawing process influences chromatin structure (Royère et al., 1991
), which may disturb decondensation of the sperm nucleus after insertion into the oocyte (Sakkas et al., 1996
). It has been reported that fertilization with cryopreserved testicular spermatozoa is inferior to that with fresh spermatozoa (De Croo et al., 1998
). In addition, a comparative study of ICSI with fresh or frozenthawed spermatozoa could demonstrate whether cryoinjury of sperm plasma membrane, which is detrimental to natural syngamy (Hammadeh et al., 1999
), can influence oocyte activation and early development of embryos obtained after ICSI.
We therefore performed a prospective, randomized study to compare the reproductive potential of fresh and frozenthawed spermatozoa from OAT patients in an ICSI procedure. The sample size of the study population was calculated to demonstrate a relative difference in pregnancy rates of 50% between both groups with a power of 80%, considering a background pregnancy rate of 30% in our institution with ICSI using a fresh sperm sample.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The patients were divided into two groups: group A where ICSI was performed with fresh spermatozoa (n = 118) and group B where ICSI was performed with frozenthawed spermatozoa (n = 122). Randomization was performed using a table of random numbers.
Ovarian stimulation and oocyte retrieval
Controlled ovarian hyperstimulation was performed using a long protocol with both long- and short-acting gonadotrophin-releasing hormone (GnRH) analogues (Decapeptyl®; Beaufor Ipsen, Destelbergen, Belgium; Diphereline®, Beaufort Ipsen; Profact®, Hoechst, Frankfurt, Germany) starting from day 21 of the preceding cycle, followed by ovarian stimulation with FSH (Metrodin®; Serono Laboratories Inc., Norwell, MA, USA) or human menopausal gonadotrophin (HMG; Pergonal®, Serono) at an individually adapted dose. About two-thirds of the patients received Decapeptyl® or Diphereline® and there was no significant difference in the repartition of the GnRH analogues among the two groups.
When the patient had at least three follicles with a diameter of 19 mm and serum oestradiol reached a concentration of 1000 pg/ml, administration of both GnRH analogue and FSH/HMG were discontinued and ovulation was induced with 10 000 IU of human chorionic gonadotrophin (HCG; Pregnyl®; Organon Inc., Canada).
Oocytes were retrieved 36 h after administration of HCG by transvaginal ultrasound-guided follicular puncture under sedation with i.v. pethidine and diazepam.
Preparation and cryopreservation of spermatozoa
Semen was collected by masturbation after 35 days of abstinence. The ejaculates were allowed to liquefy at 37°C and examined within 0.5 h of collection. The evaluation according to World Health Organisation (WHO) criteria (World Health Organization, 1992) was carried out under a light microscope. After liquefaction, semen was concentrated by centrifugation (800 g) and an aliquot from each sample was gently diluted (1:1) with freezing medium (Sigma) containing 15% of glycerol. After an equilibration period at 37°C for 10 min, the mixture was aspirated into 0.5 ml straws, which were powder-sealed. The straws were frozen according to a slow protocol, using a computer controlled freezer (Kryocell, SY-LAB, Austria) to a temperature of 150°C and then plunged into liquid nitrogen for storage. The freezing rate was 1°C/min from temperature +20°C to 7°C, then 2°C/min from 7°C to 30°C, and finally 5°C/min from 30°C to 150°C.
After 10 min, a straw of the semen was removed from the liquid nitrogen, thawed at room temperature for 15 min and examined by light microscopy. All patients in the study group had >1000 motile spermatozoa per straw.
The semen preparation for ICSI was performed as follows. An appropriate volume of both fresh or frozenthawed semen was diluted with 5 ml of Earle's medium. After centrifugation (10 min at 800 g, room temperature) the supernatant was removed and replaced with another 5 ml of Earle's medium. After a second centrifugation, the supernatant was once again removed and the pellet was overlaid with 1 ml Earle's medium and incubated (37°C, 5% CO2 in air) to separate by swim-up. For ICSI both supernatant and pellet if needed were used.
ICSI procedure and assessment of fertilization and embryonic development
Oocytes were denuded from the surrounding granulosa cells and metaphase II oocytes were microinjected as described previously (Van Steirteghem et al., 1993). Oocytes were checked for survival and fertilization 1618 h after microinjection (Nagy et al., 1994
). Fertilization was considered to be normal when two individualized or fragmented polar bodies together with two clearly visible pronuclei were present. The quality of embryos was evaluated 48 h after ICSI. Embryos were graded as good (<20% anucleate fragments), fair (2050% of anucleate fragments) and poor (>50% anucleate fragments). Embryos with <50% fragmentation were eligible for transfer.
A maximum of three embryos were transferred into the uterus on day 3 after ICSI by using a Wallace (UK), Labotect (Labotect GmbH, Germany) or Frydman (OSI, France) catheter. The Wallace and Frydman catheters were used randomly for uncomplicated transfers, the Labotec catheters were used for difficult transfers. The distribution of these three types of catheters among groups of patients was equal.
Supernumerary embryos were cultured with Vero cells and cryopreserved if they reached the blastocyst stage. The luteal phase was supported with intravaginally administered micronized progesterone, 600 mg daily (Utrogestan®, Piette, Belgium).
Pregnancy
Pregnancy was confirmed when rising serum ß-HCG concentrations were found on at least two separate occasions. Clinical pregnancy was defined by the presence of a gestational sac and fetal cardiac activity by ultrasound at ~7 weeks of pregnancy.
Statistical analysis
Continuous data analysis was performed using t-tests. Hypotheses about normal distribution were checked using the Shapiro-Wilk test. Comparisons based on contingency tables were performed using 2 test and Fisher's exact test where appropriate. All tests were two-sided. The significance level was set at 0.05. The statistical analysis was performed using SAS/STAT package.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The fertilization rate expressed as proportion of two pronuclear oocytes per successfully injected oocytes, the number of embryos obtained per cycle and the morphological score of the cleaved embryos were comparable for the two groups (Table III). Two days after ICSI, 67 and 63% of the embryos from groups A and B respectively were at the 4-cell stage (data not shown).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Few large scale studies on the fertilization and pregnancy rate after ICSI, comparing fresh and frozenthawed human spermatozoa, have been reported to date and the results differ between authors (Salom et al., 1996; Friedler et al. 1997, 1998
; Garrels et al., 1998
; Tournaye et al., 1999
; Habermann et al., 2000
). However, the majority of these studies were done with spermatozoa of testicular or epididymal origin and can therefore not be extrapolated to the results and conclusions from our study. Friedler et al. reported that no statistically significant differences were noted in fertilization rates (47 versus 44%), embryo cleavage rates (94 versus 89%), implantation rates (9 versus 11%) and clinical pregnancy rates (26 versus 27%) between ICSI cycles with fresh or cryopreserved testicular spermatozoa (Friedler et al., 1997
). Similarly, other groups (Tournaye et al., 1999
; Habermann et al. 2000
) reported that the clinical pregnancy rate per ICSI cycle was comparable between fresh and frozenthawed epididymal spermatozoa. In contrast, De Croo et al. showed in a retrospective study that implantation and live birth rates per transferred embryo are significantly lower after ICSI with frozenthawed than with fresh testicular spermatozoa (De Croo et al., 1998
).
Cryopreservation is known to impair sperm motility and to decrease fertilization rate by detrimental effects on the acrosomal structure and acrosin activity (Cross and Hanks, 1991). A flow cytometric study revealed that cryopreservation of spermatozoa affects the mitochondrial function, the membrane integrity and cell viability (Kramer et al., 1993
). The freezingthawing procedure may also be detrimental to the chromatin structure (Royère et al., 1991
; Hammadeh et al., 1999
) although a cumulative effect on sperm fertilization capacity is not definitely established. It has been proven that hidden anomalies such as higher levels of loosely packaged chromatin and damaged DNA can be present in sperm nuclei from men with deficient spermatozoa. The most frequent visible changes are related to the protamine-deficient, nicked and partially denatured DNA (Evenson et al., 1980
; Foresta et al., 1992
; Sailer et al., 1995
). Reduced binding to the human zona pellucida during IVF has been related to poor chromatin packaging and/or damaged DNA (Hosni et al., 1996).
In contrast to classical IVF, motility and the acrosome reaction are not pivotal for pregnancy outcome after ICSI. Moreover, some changes caused by cryopreservation resemble the changes occurring during natural capacitation (Gamzu et al., 1992) which may even contribute to a more efficient oocyte activation and/or pronuclear formation after ICSI. Sperm nuclei from asthenozoospermic men with nuclear abnormality decondense slowly and partially in vitro (Griveau et al., 1992
) which may contribute to failure of decondensation after ICSI (Sakkas et al., 1996
). It is important that the anomalies in the constitution of the sperm nucleus are not necessarily associated with an abnormal sperm morphology (Lolis et al., 1996
; Spano et al., 1998
). A properly performed cryopreservation (Ragni et al., 1990
) may selectively affect defective rather than normal spermatozoa (Spano et al., 1999
). This observation indicates that cryopreservation might be helpful in eliminating senescent or deficient spermatozoa that otherwise might be used as fresh spermatozoa for ICSI (Tournaye et al., 1999
). Spano et al. found that with cryopreservation and swim-up a subpopulation of spermatozoa was isolated which was characterized by a general improvement of the head morphology and motility parameters (Spano et al, 1999
). Furthermore, spermatozoa which successfully survived the freezingthawing procedure also exhibited an improved chromatin structure and nuclear maturity. These data suggest that sperm cryopreservation, when performed correctly, may not only improve fertilization rate but also enhance early embryo development parameters as well as pregnancy outcome after ICSI.
The differences in fertilization rate between fresh and frozenthawed spermatozoa were not statistically significant in this study. Similarly, we have not noticed significant differences in the time course of embryo development and embryo quality estimated 48 h after ICSI. These observations are similar to the results obtained by other authors with frozenthawed epididymal or testicular spermatozoa (Gil-Salom et al., 1996; Friedler et al., 1998
; Garrels et al., 1998
; Tournaye et al., 1999
; Habermann et al., 2000
).
In our study only 8.5% of miscarriages occurred in the group with frozenthawed spermatozoa compared with 20% in the group where fresh spermatozoa were used. Although not statistically significant this difference, if confirmed, might indicate that sperm cells which survive the freezingthawing procedure are more likely to express an intact paternal genome after fertilization. A strong paternal effect on human embryo development has indeed been demonstrated (Ron-El et al., 1991; Janny and Menezo, 1994
). Interestingly, these data coincide with those obtained after artificial insemination performed with frozen semen, where a lower incidence of spontaneous abortions and congenital malformations was observed in pregnancies obtained with frozen compared with fresh spermatozoa (Sherman, 1973
; Chong, 1985
).
In conclusion, cryopreservation of spermatozoa from men with poor sperm quality does not affect the outcome of fertilization and pregnancy rates after ICSI, within the statistical limits of this study, and fresh spermatozoa can be safely substituted by frozen ones whenever it is indicated. As far as we know, this is the first report demonstrating that cryopreserved and fresh ejaculated spermatozoa used for ICSI have at least the same reproductive potential. A larger prospective study is needed to investigate whether cryopreservation of spermatozoa before ICSI might even be helpful in selecting the most healthy sperm cells for fertilization and whether the outcome of pregnancy could therefore be improved.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cross, N.L. and Hanks, S.E. (1991) Effects of cryopreservation on human sperm acrosomes. Hum. Reprod., 6, 12791283.[Abstract]
De Croo, I., Van der Elst, J., Everaert K. et al. (1998) Fertilization, pregnancy and embryo implantation rates after ICSI with fresh or frozenthawed testicular spermatozoa. Hum. Reprod., 7, 18931897.
Devroey, P., Nagy, P., Tournaye, H. et al. (1996) Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and nonobstructive azoospermia. Hum. Reprod., 11, 10151018.[Abstract]
Evenson, D.P., Darzynkiewicz, Z. and Melamed, M.R. (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science, 240, 11311133.
Foresta, C., Zorzi, M., Rossato, M. et al. (1992) Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men. Int. J. Androl., 15, 330337.[ISI][Medline]
Friedler, S., Raziel, A., Soffer, Y. et al. (1997) Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermiaa comparative study. Fertil. Steril., 68, 892897.[ISI][Medline]
Friedler, S., Raziel, A., Soffer, Y. et al. (1998) The outcome of intracytoplasmic injection of fresh and cryopreserved epididymal spermatozoa from patients with obstructive azoosperniaa comparative study. Hum. Reprod., 13, 18721877.[Abstract]
Gamzu, R., Yogev, L., Yavetz, H. et al. (1992) Fresh and frozenthawed human sperm bind in a similar pattern to the zona pellucida in the hemizona assay. Fertil. Steril., 58, 12541256.[ISI][Medline]
Garrels, K.L., Zini, A., Casper, R.F. et al. (1998) Fresh and frozen epididymal sperm yield comparable pregnancy rates for intracytoplasmic sperm injection. Arch. Androl., 41, 159165.[ISI][Medline]
Gil-Salom, M., Romero, J., Mingues, Y. et al. (1996) Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum. Reprod., 11, 13091313.[Abstract]
Griveau, J.F., Charbonneau, M., Blanchard, Y. et al. (1992) Decondensation of human sperm nuclei and HP1 protamine degradation from normosermia and asthenospermia in Xenopus egg extracts. Arch. Androl., 29, 127136.[ISI][Medline]
Habermann, H., Seo, R., Cieslak, J. et al. (2000) In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozenthawed testicular spermatozoa. Fertil. Steril., 73, 955960.[ISI][Medline]
Hammadeh, M.E., Askari, A.S., Georg, T. et al. (1999) Effect of freezethawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int. J. Androl., 22, 155162.[ISI][Medline]
Hoshi, K., Katayose, H., Yanagida, K. et al. (1996) The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil. Steril., 66, 634639.[ISI][Medline]
Janny, L. and Ménézo Y.J. (1994) Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38, 3642.[ISI][Medline]
Kramer, R.Y., Garner, D.L., Bruns, E.S. et al. (1993) Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa. J. Androl., 14, 374384.[Abstract]
Lolis, D., Georiou, I., Syrrou, M. et al. (1996) Chromomycin A3 staining as an indicator of protamine deficiency and fertilization. Int. J. Androl., 19, 2327.[ISI][Medline]
Nagy, Z.P., Liu, J., Jorris, H. et al. (1994) Time course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplsmic sperm injection. Hum. Reprod., 9, 17431748.[Abstract]
Ng, S.C., Bongso, A. and Ratnam, S. (1991) Microinjection of human oocytes: a technique for severe oligoasthenoteratozoospermia. Fertil. Steril., 56, 11171123.[ISI][Medline]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Ragni, G., Caccamo, A.M., Dalla Serra, A. et al. (1990) Computerized slow-stage freezing of semen from men with testicular tumors of Hodgkin's disease preserves sperm better than standard vapor freezing. Fertil. Steril., 53, 10721075.[ISI][Medline]
Ron-El, R., Nachum, H., Herman, A. et al. (1991) Delayed fertilization and poor embryonic development associated with impaired semen quality. Fertil. Steril., 55, 338344.[ISI][Medline]
Royère, D., Hamamah, S., Nicolle, J.C. et al. (1991) Chromatin alteration induced by freeze thawing influence the fertilizing ability of human sperm. Int. J. Androl., 14, 328332.[ISI][Medline]
Sailer, B.L., Jost, L.K. and Evenson, D.P. (1995) Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J. Androl., 16, 8087.
Sanger, W.G., Olson, J.H. and Sherman, J.K. (1992) Semen cryobanking for men with cancercriteria change. Fertil. Steril., 58, 10241027.[ISI][Medline]
Sakkas, D., Urner, E., Bianchi, P.G. et al. (1996) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum. Reprod., 11, 837843.[Abstract]
Sherman, J.K. (1973) Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil.Steril., 24, 397412.[ISI][Medline]
Spano, M., Kolstad, H., Larsen, S.B. et al. (1998) The applicability of the flow cytometric sperm structure chromatin assay in epidemiological studies. Hum. Reprod., 13, 24952505.[Abstract]
Spano, M., Cordelli, E., Leter G. et al. (1999) Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. Mol. Hum. Reprod., 5, 2937.
Tournaye, H., Devroey, P., Liu, J. et al. (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil. Steril., 61, 10451050.[ISI][Medline]
Tournaye, H., Merdad, T., Silber, S. et al. (1999) No differences in outcome after intracytoplasmic sperm injection with fresh or with frozenthawed epididymal spermatozoa. Hum. Reprod., 14, 9095.
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm insemination. Hum. Reprod., 8, 10611066.[Abstract]
World Health Organization (1992) WHO laboratory manual for the examination of human semen and spermcervical mucus interaction. 3rd edn, Cambridge University Press, Cambridge.
Submitted on November 27, 2000; accepted on July 2, 2001.