| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Case Report |
,
From the * Laboratoire de Biologie de la
Reproduction, SIHCUS–CMCO, Schiltigheim, France; the
Laboratoire de Biologie de la Reproduction,
Hôpital Jeanne de Flandre, Lille, France; the
Institut de Génétique et de
Biologie Moléculaire et Cellulaire, Illkirch, France; the
Faculté de Médecine, Laboratoire
d'Histologie et d'Embryologie, Faculté de Médecine, Lille,
France; and the || Service d'Andrologie,
Hôpital Calmette, Lille, France.
| Correspondence to: I. Koscinski, Laboratoire de Biologie de la Reproduction, SIHCUS–CMCO, 19 rue Louis Pasteur, 67303 Schiltigheim cedex (e-mail: isabelle.koscinski{at}chru-strasbourg.fr). |
| Received for publication March 21, 2006; accepted for publication September 18, 2006. |
A safety pool of frozen sperm could be constituted for only 45 of the patients. For the last 4 (P1, P2, P3, P4), all spermatozoa were dead after cryopreservation. To avoid TESE in the event of an absence of spermatozoa on the day of the ICSI, we proposed the use of sperm ejaculated the day before the ICSI. We report here the results of the ICSI for these 4 cases that used spermatozoa ejaculated on the previous day.
Materials and Methods
Semen examination, cryopreservation procedure, and survival test—
After 3–4 days of sexual abstinence, all cryptozoospermic patients
were asked to produce semen twice in an interval of 2 hours. Freshly
ejaculated semen was mixed with 5 volumes of IVF culture medium (IVF 30;
Vitrolife, Gothenburg, Sweden) and centrifuged at 300 x g for
10 minutes. The pellet was gently resuspended in 50–300 µL of culture
medium, depending on pellet size. Twenty percent of the suspension was then
spread in 5-µL droplets on a culture dish, covered with mineral oil,
incubated 10 minutes at 37°C (5% CO2), and examined under an
inverted phase contrast microscope. Motile or living sperm were identified by
the single-sperm curling test (SSCT; Ahmadi
and Ng, 1997), were subsequently counted for each droplet, and
were placed, with the use of an injection micropipette, into a second drop of
culture media—the "survival droplet"—for the survival
test. When at least 1 motile or live sperm could be detected, the remaining
sperm preparation was frozen. This process was repeated until sperm could be
observed or the suspension was
finished.
|
For freezing, the sperm preparation was mixed at room temperature with an equal volume of Freezing Medium (TEST Yolk Buffer; Irvine Scientific, Santa Ana, Calif), and 30–50 µL of this suspension was loaded into straws and subjected to a standard cooling program (–10°C/min from room temperature to –7°C, then –20°C/min to –70°C; immersion into liquid nitrogen) in a controlled temperature freezer (Nicool LM10; AirLiquide, Marne la Vallée, France). As a test, 1 straw was thawed for 2 minutes at 37°C. Its contents were diluted 5 times very slowly with IVF 30, incubated at 37°C (5% CO2) for 10 minutes, and centrifuged at 300 x g for 10 minutes. The pellet, after resuspension in 30–50 µL of IVF medium, was spread in 5-µL droplets and examined as previously described to detect motile sperm cells. When fewer than 10 motile spermatozoa were observed, a SSCT was performed with the motionless spermatozoa. At least 10 motile or living thawed spermatozoa were required for a successful "safety pool." When this number was not reached, the procedure was repeated a maximum of 6 times in the next months, or until a minimum of 10 live spermatozoa was obtained. If this minimum was obtained, the safety pool was considered an alternative to freshly ejaculated sperm in the event of transient azoospermia on the day of the ICSI. When this minimal pool of 10 frozen spermatozoa could not be identified, the treatment depended on the survival test as described below.
Survival test— For all cryptozoospermic patients, the survival test consisted of examining the cultured survival droplet after 1 day for sperm motility and vitality (SSCT). The survival test of 1-day-old sperm was performed without motility enhancer at the same time as sperm examination for the cryopreservation assay (up to 7 times, but an average of 3 times before the ICSI attempt and with a last assay 2 weeks before the ICSI attempt). If at each examination at least 10 motile/alive spermatozoa could be found in the survey droplet, the survival test was considered positive, and 1-day-old sperm was considered an alternative to freshly ejaculated sperm. In the absence of any safety pool of ejaculated sperm (either frozen or incubated), a testicular biopsy was used to make a safety pool of frozen testicular spermatozoa. Thus, the ICSI would be performed with ejaculated sperm (produced twice on the morning of the attempt), which if necessary would be supplemented, in order of preference, with cryopreserved sperm, sperm cultured for 24 hours, and, finally, frozen testicular spermatozoa (Figure).
For 45 of 49 patients, a safety pool of frozen sperm was successfully obtained; for these patients, the survival test was also positive. For practical reason, and to optimize sperm production the day of the ICSI with several days of sexual abstinence, we planned to use freshly ejaculated sperm from these patients for the ICSI, although frozen spermatozoa could be used in case of insufficient sperm count (or absence). For the 4 others, despite up to 5 attempts at cryopreservation, creation of safety pool of frozen sperm was not possible: spermatozoa were observed and cryopreservation performed, but after thawing/rehydration all spermatozoa were dead (SSCT). In contrast, the survival test was regularly positive. For these 4 patients, the use of spermatozoa cultured for 1 day was proposed as an alternative to freshly ejaculated spermatozoa in the event of azoospermia the day of the ICSI.
We report here the ICSI results for these 4 patients, using spermatozoa cultured for 1 day. Duration of infertility, male and female age and pathology, testis size, male seric FSH and testosterone levels, as well as karyotype and results of a search for Y microdeletions are summarized in Table 1. Even if testis size was low in all cases, FSH was abnormally elevated in only 1 patient. All 4 patients regularly had sperm counts under 50 spermatozoa per ejaculate, with at least 1 episode of transient azoospermia.
|
The day before, as well as the day of, oocyte retrieval, patients were asked to produce semen twice in 2 hours; this semen was then treated as described above. Because no spermatozoa were in the ejaculates on the morning of the ICSI, spermatozoa from the previous day were used for ICSI.
Ovarian stimulation, oocyte retrieval, and ICSI procedure were performed according to standard practices (Van Steirteghem et al, 1993; Rienzi et al, 1998).
Spermatozoa injected were selected according to their motility and morphology. All had either progressive or nonprogressive motility, without any use of motility enhancers. Morphology was assessed under an inverted microscope (400x). Most spermatozoa met our criteria: no angulation, normal head size, and a maximum of only 1 vacuole. Spermatozoa with cytoplasmic rest on the first day were very often motionless by the second day. The assessment of fertilization, embryonic cleavage, and morphologic quality were performed according to standard practices (Ron-El et al, 1997). Embryo transfers occurred 70 hours after ICSI and a good quality embryo was defined as having at least 3 blastomeres on the second day or 6 on the third day, with less than 20% of anucleated fragments. No more than 3 embryos were transferred, and routine progestational luteal support was given. Clinical pregnancies were defined as a sonographic visualization of at least 1 gestational sac.
Results
ICSI results are summarized in Table
2. For all 4 patients, neither ejaculate produced on the day of
the oocyte retrieval contained spermatozoa. Thus, spermatozoa of the previous
day were employed for ICSI; the count of spermatozoa available ranged from 10
to 32 (mean ± SD, 18 ± 10). The fertilization rates ranged from
27% to 75% (mean 41%). Table 3
summarizes the characteristics (morphology and motility) of spermatozoa used
for successful fertilizations: in most cases, zygotes resulted from
spermatozoa with normal morphology and progressive motility. In the 4
attempts, the cleavage rates were good (mean 92%), and 4 out of 5 embryos were
of "good quality" for patient P1, and 2 out of 2 for P3 and P4.
For P2 however, all 3 embryos were of poor quality. Nevertheless, in keeping
with the patients' wishes, an embryo transfer took place for each of the
couples. For the 2 patients in which fertilization rates were highest (50% for
P1 and 75% for P4), the transfer of good quality embryos resulted in
evolutionary pregnancies. In the 2 other cases (P2 and P3), in which the
fertilization rate was around 25%, the proportion of good quality embryos was
low, and no pregnancy occurred. It is important to note that the poor quality
embryos for P2 could have been due to the woman's pathology (polycystic
ovarian syndrome). For P1 and P4, 2 healthy children were born. For P1, the
transfer of a second frozen embryo led to a second healthy child.
|
|
Discussion
In the case of cryptozoospermia, fluctuations in sperm number can lead to
the absence of sperm in the ejaculate on the day of oocyte retrieval. In this
case, testicular spermatozoa are often used as an alternative to ejaculated
ones. But, in patients with severe primary testicular defect and likely only
very focalized spermatogenesis, testicular biopsy can fail to provide
spermatozoa (Ron-El et al,
1997), and some authors suggest the TESE be repeated when negative
to enhance sperm retrieval (Vernaeve et
al, 2006). Moreover, in addition to potential andrological
defects, testicular biopsies can involve the risk of nonmature gametes. To
have the greatest possible chance of using ejaculated sperm for ICSI attempts,
to have the greatest possible chance of using ejaculated sperm from patients
with cryptozoospermia, our center proposes a strategy including the creation
of 2 safety pools of spermatozoa: 1 frozen and 1 collected the day before ICSI
and cultured overnight. In the event that the cryopreserved sperm are
nonviable, we propose as an alternative the use of sperm ejaculated 1 day
earlier instead freshly ejaculated sperm.
In the cases presented here, patients' ejaculates regularly contained fewer than 50 sperm. They agreed to come to the center at least 3 times in the months before the ICSI attempt to check the survival of their sperm during a 24-hour period of culture. Survival was regularly more than 50%, and in all cases, at least 10 spermatozoa were motile after the 24-hour incubation. They declared, retrospectively, that this procedure permitted them to approach the ICSI day without fear of the absence of spermatozoa on the day of oocyte retrieval.
Interestingly, bacterial contamination was rarely a problem. In more than 200 survival tests for 49 patients, we recorded only 3 instances of contamination. This was likely because spermatozoa were, in effect, washed twice: they were centrifuged, resuspended in sterile medium, then isolated and transferred to fresh, sterile medium with an injection micropipette.
On the day before oocyte retrieval, the number of motile (or living) sperm was very low, especially for 2 patients, P1 and P4, who obtained a child. Not surprisingly, we observed that zygotes came most often from spermatozoa with apparently normal shape (assessed with a standard inverted microscope at 400x magnification; Table 3) and progressive motility and that pregnancies occurred for the patients with the 2 best fertilization rates. Fertilization rates (FR, 41%), cleavage rates (CR, 92%), and the proportion of good quality embryos (GE, 67%) were similar to those observed in our center with freshly ejaculated sperm of cryptozoospermic patients (n = 40: FR 49%, CR 95%, GE 43%, pregnancy/delivery rate 12%, 5 deliveries) or with safety pools of frozen spermatozoa (n = 5: FR 54%, CR 92%, GE 33%, 1 child). Over the same period, our center found that these ICSI results were not significantly different of those obtained using TESE-testicular spermatozoa for patients with nonobstructive azoospermia (n = 42; FR 30%, CR 95%, GE 50%, pregnancy/delivery rate 19%, 8 deliveries). For Ron-El et al (1997), too, the results of ICSI with ESP fresh-ejaculated sperm (FR 41%, CR 88%, GE 56%) are very similar to those with testicular sperm (FR 49%, CR 93%, GE 58%) in cryptozoospermic men, and both are somewhat lower than with ejaculated spermatozoa in the regular ICSI program.
In the 4 cases reported here, the use of ejaculated sperm cultured for 24 hours rather than TESE spermatozoa resulted in ICSI parameters similar to those of other cryptozoospermic patients using fresh or frozen sperm or with nonobstructive azoospermic patients using frozen testicular spermatozoa. The incubation of spermatozoa for 24 hours in culture medium does not seem to have impaired their fertility. This was not entirely expected, considering that others have observed a decrease in the quality of ejaculated sperm cultured for 24 hours (but after selection by swim-up technique; Calamera et al, 2001) or testicular sperm (Dalzell et al, 2004). In ejaculated and cultured sperm, the major alteration observed during 24 hours of in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. Importantly, however, no substantial differences in sperm viability or chromatin condensation were observed (Calamera et al, 2001); this is in contrast to immature testicular spermatozoa, in which increased DNA fragmentation was observed and probably linked to a greater vulnerability to environment (Dalzell et al, 2003). Muratori et al (2000, 2003) suggested the involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation; they found that DNA fragmentation rate was increased in teratozoospermic samples, particularly in the case of persistent cytoplasmic residues, which are indicative of immature spermatozoa. Still, it is not possible to demonstrate that the risk of DNA damage is not increased in these patients. Indeed, with a severe defect in spermatogenesis, a significant proportion of spermatozoa injected into oocytes can contain damaged DNA. Injection of oocytes with spermatozoa containing abnormal chromatin would probably result in a failure of fertilization; this could explain the poor fertilization rates in cryptozoospermic patients, although Twigg et al (1998) demonstrated that chromatin-damaged spermatozoa can result in a zygote, and Gandini et al (2004) reported full-term pregnancies achieved by ICSI despite high levels of sperm chromatin damage. Today, it appears difficult to tell whether the use of cultured cryptozoospermic spermatozoa is more or less safe than the use of frozen/thawed testicular spermatozoa. Large-scale studies are necessary to determine the effects of sperm DNA damage on the outcome of ART, especially on children's health.
Conclusion
The use of ejaculated sperm cultured for 24 hours can help avoid the need
both for an invasive procedure like TESE and for the use of immature
testicular spermatozoa. Considering the ICSI results of the 4 cases reported
here, this method could be an alternative to freshly ejaculated sperm for
cryptozoospermia patients with azoospermia on the day of ICSI. Finally, it
improves the psychological state of cryptozoospermic patients who feel much
stress and anguish in the absence of a safety pool of frozen sperm. Today,
whereas reproductive medicine tries to treat each patient couple on a
case-by-case basis in hopes of maximizing the chance of conception while
minimizing the risks and discomfort, the 4 cases described here provide an
interesting avenue of treatment.
Acknowledgments
The authors thank L. Acitokahn and MacLean Sellars for language help.
References
Ahmadi A, Ng SC. The single sperm curling test, a modified hypoosmotic swelling test, as a potential technique for the selection of viable sperm for intracytoplasmic sperm injection. Fertil Steril. 1997;68: 346 –50.[CrossRef][Medline]
Ariel M, Cedar H, McCarrey J. Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis. Nat Genet. 1994;7: 59 –63.[Medline]
Barri PN, Vendrell JM, Martinez F, Coroleu B, Aran B, Veiga A. Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients. Reprod Biomed Online. 2005; 10: 735 –739.[Medline]
Calamera JC, Fernandez PJ, Buffone MG, Acosta AA, Doncel GF. Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect. Andrologia. 2001; 33: 79 –86.[CrossRef][Medline]
Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SE. Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril. 2004; 82: 1443 –1445.[CrossRef][Medline]
Dalzell LH, Thompson-Cree ME, McClure N, Traub AI, Lewis SE. Effects of 24-hour incubation after freeze-thawing on DNA fragmentation of testicular sperm from infertile and fertile men. Fertil Steril. 2003;79: 1670 –1672.
Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D,
Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm
aspiration compared with testicular sperm extraction by open biopsy in men
with non-obstructive azoospermia. Hum Reprod. 1997; 12: 1488
–1493.
Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G,
Ciriminna R, Culasso F, Dondero F, Lenzi A, Spano M. Full-term pregnancies
achieved with ICSI despite high levels of sperm chromatin damage.
Hum Reprod. 2004; 19: 1409
–1417.
Harrington TG, Schauer D, Gilbert BR. Percutaneous testis biopsy: an alternative to open testicular biopsy in the evaluation of the subfertile man. J Urol. 1996; 156: 1647 –1651.[CrossRef][Medline]
Kerjean A, Dupont JM, Vasseur C, Le Tessier D, Cuisset L, Paldi A,
Jouannet P, Jeanpierre M. Establishment of the paternal methylation imprint of
the human H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol
Genet. 2000;9: 2183
–2187.
Manning M, Junemann KP, Alken P. Decrease in testosterone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men. Lancet. 1998; 352: 37 .[CrossRef][Medline]
Muratori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E.
Spontaneous DNA fragmentation in swim-up selected human spermatozoa during
long term incubation. J Androl. 2003; 24: 253
–262.
Muratori M, Piomboni P, Baldi E, Filimberti E, Pecchioli P, Moretti E, Gambera L, Baccetti B, Biagiotti R, Forti G, Maggi M. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 2000;21: 903 –912.[Abstract]
Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation
of human oocytes may improve fertilization and embryo quality after
intracytoplasmic sperm injection. Hum Reprod. 1998; 13: 1014
–1019.
Ron-El R, Strassburger D, Friedler S, Komarovski D, Bern O, Soffer
Y, Raziel A. Extended sperm preparation: an alternative to testicular sperm
extraction in non-obstructive azoospermia. Hum Reprod. 1997; 12: 1222
–1226.
Schill T, Bals-Pratsch M, Kupker W, Sandmann J, Johannisson R, Diedrich K. Clinical and endocrine follow-up of patients after testicular sperm extraction. Fertil Steril. 2003; 79: 281 –286.[CrossRef][Medline]
Schlegel PN, Su LM. Physiological consequences of testicular sperm
extraction. Hum Reprod. 1997; 12: 1688
–1692.
Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human
spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm
injection. Hum Reprod. 1998; 13: 1864
–1871.
Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J,
Wisanto A. high fertilization rate after intracytoplasmic sperm injection.
Hum Reprod. 1993; 8: 1061
–1066.
Vernaeve V, Verheyen G, Goossens A, Van Steirteghem A, Devroey P,
Tournaye H. How successful is repeat testicular sperm extraction in patients
with azoospermia? Hum Reprod. 2006; 21: 1551
–1554.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
