Skip to main content

Impact on ICSI outcomes of adding 24 h of in vitro culture before testicular sperm freezing: a retrospective study



To compare sperm parameters and intracytoplasmic sperm injection (ICSI) outcomes for testicular spermatozoa frozen on the day of the biopsy (DO) with those frozen after 24 h of in vitro culture (D1).


In this retrospective study, from 1999 to 2012, forty-nine azoospermic patients were included to compare sperm (motility and viability) and outcomes (fertilization (FR), implantation (IR), pregnancy (PR) and delivery rates (DR)).


The in vitro culture increased total motility (+2.8 %, p = 0.0161) but decreased viability (−8.3 %, p = 0.007). After 24 h of culture, the post-thaw changes in motility and viability were not significant. Twenty-six couples underwent ICSI: thirty–four ICSI were performed with spermatozoa cryopreserved at D0 and eighteen with spermatozoa frozen at D1. Cumulated IR and DR were lower for ICSI with D1 spermatozoa than with D0 spermatozoa (IR: 21.6 % with D0 vs. 9.8 % with D1, p = 0.102; DR: 27.5 % with D0 vs. 8.3 % with D1, p = 0.049).


Despite improving motility, freezing spermatozoa 24 h after testicular biopsy had a potential negative effect on ICSI outcomes, notably on delivery rates. These results may be related to the detrimental impact of the additional culture on the nuclear integrity of sperm.



Comparer les paramètres spermatiques et les issues de fécondation in vitro avec micro-injection (ICSI) de spermatozoïdes testiculaires congelés le jour de la biopsie (D0) avec ceux congelés après 24 heures de culture in vitro (D1).


Dans cette étude rétrospective, de 1999 à 2012, quarante-neuf patients présentant une azoospermie ont été inclus pour comparer les paramètres spermatiques (mobilité et vitalité) et les issues d’ICSI (taux de fécondation (FR), d’implantation (IR), de grossesse (PR), et d’accouchement (DR)).


La culture in vitro augmentait la mobilité (+2.8 %, p = 0.0161) mais diminuait la vitalité (-8.3 %, p = 0.007). Après cumul des 24 heures de culture et congélation, les différences observées n’étaient plus significatives. Vingt-six couples ont eu au moins une ICSI : 34 ont été réalisées avec des spermatozoïdes congelés à D0 et 18 ont été réalisées avec des spermatozoïdes congelés à D1. Les taux d’implantation et d’accouchement cumulés étaient plus faibles avec les spermatozoïdes congelés à D1 par rapport à ceux congelés à D0 (IR: 21.6 % avec D0 vs. 9.8 % avec D1, p = 0.102; DR: 27.5 % avec D0 vs. 8.3 % avec D1, p = 0.049).


Malgré l’augmentation de la mobilité, la congélation de spermatozoïdes testiculaires 24 heures après la biopsie apparait avoir un impact négatif sur les issues d’ICSI, notamment sur les taux d’accouchement. Ces résultats pourraient être en lien avec les effets néfastes de l’association des deux procédés (l’incubation pendant 24H cumulée à la congélation-décongélation) sur l’intégrité nucléaire spermatique.


Testicular sperm extraction (TESE) and ICSI have become common procedures in assisted reproduction programs for the treatment of both obstructive and non-obstructive azoospermia. As similar results in terms of fertilization, pregnancy and delivery rates are observed with the use of both frozen and fresh testicular spermatozoa [1], most centers freeze testicular spermatozoa retrieved after TESE and perform asynchronous ICSI. Indeed, this strategy makes it possible to prevent ICSI cancellations if no spermatozoa were retrieved [2].

ICSI achieved with testicular spermatozoa is acutely dependent on spermatozoa viability. However, their motility is often poor, making the selection of viable spermatozoa difficult. Therefore, several complementary approaches to sperm selection have been developed in order to detect immotile but live sperm or to optimize sperm function in vitro [310]. Among these is the ability of live sperm to respond to a hypoosmotic test, in which live sperm show swelling of the tail in hypoosmotic media [5]. A variety of substances, such as pentoxifylline, are also commonly used to stimulate sperm motility [3, 6, 810]. Some authors also advise promoting sperm motility by increasing the culture time as an alternative to viability tests. Indeed, in 1995, Craft and colleagues reported in the Lancet a significant increase in the number of progressively motile sperm after 24–72 h of in vitro culture in in vitro fertilization culture medium [11]. A few years later, other studies praised this practice [1222]. Indeed, it has been reported that extended in vitro culture improves the motility of testicular sperm [1220] with a maximum motility rate between 48 and 72 h of in vitro culture [17, 19, 20]. Based on these results, numerous teams around the world, like the previous head of Dijon’s Laboratory in 1999, developed this practice in laboratories. However, to date, only two studies on fresh cultured testicular spermatozoa have reported data on delivery rates [23, 24]. Moreover, there are currently no data available concerning the performance of ICSI using testicular spermatozoa frozen after an additional in vitro culture time of 24 h. In addition, some results in the literature suggest that prolonged in vitro incubation may have negative effects on nuclear status, in particular a deterioration in the integrity of sperm nuclei, which may potentially lead to adverse ICSI outcomes [25, 26].

Therefore, the aim of the present retrospective study was to analyze the interest and impact of an additional incubation period of 24 h followed by cryopreservation of testicular spermatozoa. For these purposes, we analyzed the testicular sperm parameters and we investigated ICSI outcomes associated with using testicular spermatozoa cryopreserved on the same day of retrieval or following 24 h of culture.



In this retrospective study, only patients with successful testicular sperm extraction (TESE) and with two cryopreserved sperm samples (one on the day of the testicular sperm retrieval (D0) and one the following day, after 24 h of supplementary in vitro culture (D1)) between January 1999 and December 2012 at Dijon University Hospital (France) were included.

During this period, 178 patients with obstructive (OA) and non-obstructive azoospermia (NOA) underwent testicular sperm extraction (TESE) at our center. Testicular sperm samples were successfully retrieved from 106 patients (59.6 %). For forty-nine (46.2 %) of these 106 patients, testicular sperm samples were cryopreserved at D0 and D1. Thus, semen analyses were based on 49 patients, 7 had NOA (14.3 %) and 42 had OA (85.7 %).

ICSI was carried out in 26 of the patients to study their outcomes: spermatozoa frozen at D0 (SPZ D0) was used for 17 patients, spermatozoa frozen at D1 (SPZ D1) was used for 5 patients and both testicular spermatozoa frozen at D0 and D1 was used for 4 patients on different ICSI cycles (identified as “the subgroup of same couples”). Of the 26 patients, 5 had NOA (19.2 %) and 21 had OA (80.8 %). The choice to use spermatozoa cryopreserved at D0 or D1 was based on the results of post-thawing tests.

Institutional Review Board approval was obtained for the collection of data of couples who had undergone ICSI cycles.

Testicular sperm extraction and cryopreservation

The biopsies were obtained during open surgery under general anesthesia. Two testicular specimens of approximately 5 mm long and 5 mm thick were obtained from two different locations on the same testis. The biopsy samples were then independently minced using sterile needles in a dish containing 3 ml of Ferticult Hepes (Fertipro, Belgium) and incubated at 37 °C under 5.5 % CO2 and a humidified atmosphere for 30 min in IVF medium (Fertipro, Belgium) [27]. Each supernatant was then transferred to a 15 ml conical Falcon tube and diluted with a sperm cryoprotectant medium (SpermFreeze, Fertipro, Belgium) according to the manufacturer’s protocol. This semen sample was cryopreserved in freezing straws (Cryo Bio Systems, France). After being maintained at room temperature for 10 min, the straws were slowly cooled in liquid nitrogen vapour as described by Karacan et al. [24], and stored in liquid nitrogen at −196 °C.

After the addition of IVF medium (3 ml) to the remaining minced tissue, the culture was pursued for an additional 24 h in the same conditions. The next day (D1), the second supernatant was frozen using the same protocol as for the D0 sample.

The thawing test was performed the same day for both testicular sperm suspensions cryopreserved at D0 and D1 as follows: the straws were taken out of the liquid nitrogen and kept at room temperature (RT) for 8 min before draining into a test tube. Ten microliters of fresh and frozen-thawed testicular sperm at D0 and D1 were analyzed for concentration, motility, and viability using eosin-nigrosin smears [28].

ICSI outcomes

ICSI processes

The female partners of these 26 patients underwent ovarian stimulation. Cryopreserved testicular spermatozoa were thawed on the same day as oocyte retrieval only if the presence of mature oocytes was confirmed after oocyte denudation. After thawing, the testicular spermatozoa were washed in 1 ml of culture medium (Ferticult flushing, Fertipro, Belgium) with centrifugation at 600 g for 5 min at RT. After centrifugation, the supernatant was removed and the pellet was resuspended in about 50 μL of culture medium with Hepes (Ferticult Hepes, Fertipro, Belgium). Then assisted fertilization by ICSI was performed as previously described [29].

ICSI evaluation

Fertilization was assessed 17–19 h after ICSI by checking the number of pronuclei. The fertilization rate was defined as the ratio between the number of diploid zygotes and the number of mature oocytes. Embryo cleavage was evaluated after 44 +/−1 h of culture. The morphological appearance of embryos was monitored according to the number and the size of the blastomeres (regular or irregular cleavage) as well as the percentage of anucleate fragments [30]. Embryos seen fertilized at day 1 with regular 4- to 5-cell embryos at day 2 with less than 20 % fragmentation and without any multinuclear blastomeres were regarded as “TOP” grade. The percentage of TOP embryos was defined as the ratio between the number of TOP embryos and the total number of embryos. Depending on the age of the women, the number of previous cycles, and the number and quality of embryos available, 1 or 2 embryos were transferred at either day 2 or day 3 after oocyte retrieval. Progesterone was administered vaginally every day from the day of oocyte retrieval until the time of a negative pregnancy test or until 8 weeks of gestation. Embryo cryopreservation and frozen-thawed embryo transfer were performed as previously described [31].

Clinical pregnancy was determined by the presence of an intrauterine gestational sac with a fetal heartbeat on ultrasound examination 4–5 weeks after the embryo transfer. The implantation rate was the ratio between the number of gestational sacs and the number of transferred embryos. The delivery rate was the ratio between the number of deliveries and the number of embryo transfers. In the same way, the outcomes of frozen embryo cycles were also analyzed.

Statistical analysis

Quantitative variables were described using means and standard deviations, qualitative variables using frequencies and proportions. Sperm parameters before and after freezing-thawing as well as before and after the additional in vitro culture of 24 h were compared by means of Wilcoxon’s paired test.

The results for fresh embryo transfers alone or cumulated with those of frozen embryo transfers between spermatozoa cryopreserved at D0 and D1 were compared using the Chi-square test (or Fisher’s exact test, if the expected values were < 5). Data were analyzed using SAS version 9.2 in the Clinical Research Unity of the University Hospital of Dijon. Differences were considered significant when p < 0.05.


Semen analysis

The results for the effects of freezing and/or in vitro culture on sperm parameters are presented in Table 1. Overall, freezing decreased the motility of spermatozoa cryopreserved at D0 or D1 (–6.0 % and –6.3 %, respectively; p < 0.001) and also their viability (–25.2 % and –20.8 %, respectively; p < 0.001). The in vitro culture increased total motility (+2.8 %, p = 0.0161) but decreased viability (–8.3 %, p = 0.007). This increase in motility after 24 h of supplemental in vitro culture was no longer significant after thawing (+1.4 %, p = 0.0895). Identical effects were found for testicular spermatozoa from NOA and OA patients. The mean number of frozen straws was 14.6 +/–8.5 (range: 3 to 34 straws) and 8.2 +/–4.5 (range: 2 to 23 straws) at D0 and D1, respectively.

Table 1 Effects of in vitro culture and freezing on sperm parameters

ICSI outcomes

Among the 49 patients included to study their sperm parameters, twenty-six underwent ICSI. Thirty–four ICSI were performed with spermatozoa cryopreserved at D0. Eighteen ICSI were performed with spermatozoa frozen at D1. Finally, for 4 couples, 12 ICSI treatments were done with both spermatozoa cryopreserved at D0 (4 ICSI) and D1 (8 ICSI), on different cycles.

The ICSI results obtained with spermatozoa frozen at D1 were compared with those obtained with spermatozoa frozen at D0 (Table 2).

Table 2 Baseline characteristics and ICSI outcomes

For the same couples, fertilization, embryo cleavage and frozen embryo rates were significantly lower when spermatozoa frozen at D1 were used than was the case with spermatozoa frozen at D0 (Table 2). In the same couples as well as in all couples, the implantation, pregnancy and delivery rates were lower when spermatozoa frozen at D1 were used than when spermatozoa frozen at D0 were used. Among all couples, the delivery rate was significantly higher with spermatozoa frozen at D0 than with spermatozoa frozen at D1 (29.4 % (D0) vs. 5.6 % (D1), p = 0.045). No difference was found according to the type of azoospermia (OA vs NOA–data not shown). The cumulated delivery rates taking into account fresh and frozen embryo transfers were significantly higher for spermatozoa frozen at D0 (27.5 % (D0) vs. 8.3 % (D1), p = 0.049).


To the best of our knowledge, no study has assessed the impact on ICSI outcomes of an additional 24 h of in vitro culture before testicular sperm freezing. This study revealed that the best outcomes were achieved when the testicular sperm was frozen on the day of the testicular biopsy. Despite improving motility, the additional 24 h of culture before freezing had a negative effect on fertilization and implantation.

Our results confirmed that the in vitro culture of testicular spermatozoa for 24 h significantly improved their motility [1120]. However, the increase in motility was no longer significant after thawing. Emiliani and collaborators [15] observed similar results when the testicular spermatozoa were cultured after thawing. In addition, as previously reported, we found that the freezing step decreased testicular sperm viability [27]. Thus, even though the second freezing step performed after 24 h of in vitro culture could increase the number of straws available for further ICSI attempts, the accumulation of both sperm treatment processes may be unfavorable to sperm quality/nuclear integrity.

In the current study, even though the number of ICSI cycles was relatively small, the significant results proved that the additional 24 h of culture followed by the freezing step had adverse effects on ICSI outcomes. Indeed, as highlighted in ICSI in the same couples, the fertilization rate was lower with testicular spermatozoa frozen one day after the biopsy than was the case with spermatozoa frozen immediately after the biopsy. To date, there are no data available concerning the efficacy of ICSI carried out with cultured and then frozen testicular spermatozoa. However, some authors have reported ICSI outcomes after testicular sperm cryopreservation alone. Although fertilization and pregnancy rates were similar [32], implantation rates were lower [1] and spontaneous abortion rates were higher [33] in ICSI cycles with frozen testicular spermatozoa than with fresh spermatozoa. Moreover, even though several authors have reported that extended in vitro culture improved the motility of testicular spermatozoa, very few studies have analyzed implantation, pregnancy and delivery rates following this treatment process [18, 23, 24, 34] (Table 3). Only one reported a significant improvement in the fertilization rate with the use of spermatozoa incubated for 24 h [18]. However, in this study with unfrozen cultured spermatozoa, the clinical pregnancy rate was very low as compared to the biochemical pregnancy rate (35.3 % vs 53.9 %, respectively), suggesting a high number of miscarriages with spermatozoa processed in this way, which agrees with our findings. In the other study, even though the results were not significant, the delivery rates were lower in ICSI with cultured testicular spermatozoa than in ICSI without an incubation step [23]. In addition, the sperm used in ICSI pregnancies followed by miscarriage displayed high levels of fragmentation: OR = 2.68 IC95: 1.40–5.14; p = 0.003 [35]. It would thus be interesting to know if miscarriage could be explained by a high level of sperm DNA fragmentation induced by in vitro culture.

Table 3 ICSI outcomes with or without cultured testicular spermatozoa

The deleterious effects of culture on outcomes and more particularly on fertilization may be related to the detrimental impact on sperm integrity of Reactive Oxygen Species (ROS) produced by the spermatozoa themselves [36], and ROS production is known to be increased by culture [37]. In addition, even though no major changes were observed for chromatin condensation and acrosome reaction [37], severe structural chromosomal abnormalities (deletions, translocations, breaks, gaps, acentric fragments) were reported in in vitro stored ejaculated spermatozoa [38]. DNA damage assessed by the acridine-orange test on over 232 chromosome spreads from sperm samples of 2 donors revealed an excess of breaks in DNA after 24 h of in vitro culture [38]. Furthermore, DNA fragmentation measured with the TUNEL assay in testicular sperm from men with obstructive azoospermia was higher following long incubation than following short or no incubation [26]. In this latter work, the authors also reported an increase in DNA fragmentation even after the freezing step alone. Moreover, Matsuura et al. also described the influence of incubation temperature on DNA fragmentation: the temperature of 37 °C significantly increased the DNA fragmentation index compared with room temperature [39]. The addition of both procedures probably has a cumulative negative effect on sperm DNA.


In light of these significant findings, we recommend that reproductive clinics do not prolong in vitro culture before testicular sperm freezing. Indeed, by handling gametes and especially by prolonging and potentiating sperm exposure to non-physiological conditions, extended in vitro culture followed by freezing may induce iatrogenic damage, including an increase in sperm DNA fragmentation and embryo development failure.

Finally, while the implication of testicular spermatozoa in birth defects is controversial [40], we wonder if the use of in vitro cultured then frozen testicular spermatozoa could have a negative impact on the health of children thus conceived. These infants would need to be followed in the long-term to detect any resulting anomalies or other unwanted effects.



Day of the biopsy


After 24 h of in vitro culture


Deoxyribonucleic acid


Delivery rate


Fertilization rate


Intracytoplasmic sperm injection


Implantation rate


Non-obstructive azoospermia


Obstructive azoospermia


Pregnancy rate


Room temperature


Reactive oxygen species




Testicular sperm extraction


World health organization


  1. Nicopoullos JDM, Gilling-Smith C, Almeida PA, Norman-Taylor J, Grace I, Ramsay JWA. Use of surgical sperm retrieval in azoospermic men: a meta-analysis. Fertil Steril. 2004;82:691–701.

    Article  PubMed  Google Scholar 

  2. Oates RD, Lobel SM, Harris DH, Pang S, Burgess CM, Carson RS. Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa. Hum Reprod. 1996;11:133–8.

    Article  CAS  PubMed  Google Scholar 

  3. Nassar A, Mahony M, Morshedi M, Lin MH, Srisombut C, Oehninger S. Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility. Fertil Steril. 1999;71:919–23.

    Article  CAS  PubMed  Google Scholar 

  4. Nordhoff V, Schüring AN, Krallmann C, Zitzmann M, Schlatt S, Kiesel L, et al. Optimizing TESE-ICSI by laser-assisted selection of immotile spermatozoa and polarization microscopy for selection of oocytes. Andrology. 2013;1:67–74.

    Article  CAS  PubMed  Google Scholar 

  5. Verheyen G, Joris H, Crits K, Nagy Z, Tournaye H, Van Steirteghem A. Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update. 1997;3:195–203.

    Article  CAS  PubMed  Google Scholar 

  6. Aparicio NJ, de Turner EA, Schwarzstein L, Turner D. Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility. Andrologia. 1980;12:49–54.

    Article  CAS  PubMed  Google Scholar 

  7. Makler A, Makler E, Itzkovitz J, Brandes JM. Factors affecting sperm motility. IV. Incubation of human semen with caffeine, kallikrein, and other metabolically active compounds. Fertil Steril. 1980;33:624–30.

    CAS  PubMed  Google Scholar 

  8. Yovich JL. Pentoxifylline: actions and applications in assisted reproduction. Hum Reprod. 1993;8:1786–91.

    CAS  PubMed  Google Scholar 

  9. Kay VJ, Coutts JR, Robertson L. Pentoxifylline stimulates hyperactivation in human spermatozoa. Hum Reprod. 1993;8:727–31.

    CAS  PubMed  Google Scholar 

  10. Tournaye H, Janssens R, Devroey P, van Steirteghem A. The influence of pentoxifylline on motility and viability of spermatozoa from normozoospermic semen samples. Int J Androl. 1994;17:1–8.

    Article  CAS  PubMed  Google Scholar 

  11. Craft I, Tsirigotis M, Zhu JJ. In-vitro culture of testicular sperm. Lancet. 1995;346:1438.

    Article  CAS  PubMed  Google Scholar 

  12. Morris DS, Dunn RL, Schuster TG, Ohl DA, Smith GD. Ideal culture time for improvement in sperm motility from testicular sperm aspirates of men with azoospermia. J Urol. 2007;178:2087–91.

    Article  PubMed  Google Scholar 

  13. Angelopoulos T, Adler A, Krey L, Licciardi F, Noyes N, McCullough A. Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertil Steril. 1999;71:240–3.

    Article  CAS  PubMed  Google Scholar 

  14. Edirisinghe WR, Junk SM, Matson PL, Yovich JL. Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection. Hum Reprod. 1996;11:2474–6.

    Article  CAS  PubMed  Google Scholar 

  15. Emiliani S, Van den Bergh M, Vannin AS, Biramane J, Verdoodt M, Englert Y. Increased sperm motility after in-vitro culture of testicular biopsies from obstructive azoospermic patients results in better post-thaw recovery rate. Hum Reprod. 2000;15:2371–4.

    Article  CAS  PubMed  Google Scholar 

  16. Hu Y, Maxson WS, Hoffman DI, Ory SJ, Licht MR, Eager S. Clinical application of intracytoplasmic sperm injection using in vitro cultured testicular spermatozoa obtained the day before egg retrieval. Fertil Steril. 1999;72:666–9.

    Article  CAS  PubMed  Google Scholar 

  17. Liu J, Tsai YL, Katz E, Compton G, Garcia JE, Baramki TA. Outcome of in-vitro culture of fresh and frozen-thawed human testicular spermatozoa. Hum Reprod. 1997;12:1667–72.

    Article  CAS  PubMed  Google Scholar 

  18. Windt ML, Coetzee K, Kruger TF, Menkveld R, van der Merwe JP. Intracytoplasmic sperm injection with testicular spermatozoa in men with azoospermia. J Assist Reprod Genet. 2002;19:53–9.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Wu B, Wong D, Lu S, Dickstein S, Silva M, Gelety TJ. Optimal use of fresh and frozen-thawed testicular sperm for intracytoplasmic sperm injection in azoospermic patients. J Assist Reprod Genet. 2005;22:389–94.

    Article  PubMed Central  PubMed  Google Scholar 

  20. Zhu J, Tsirigotis M, Pelekanos M, Craft I. In-vitro maturation of human testicular spermatozoa. Hum Reprod. 1996;11:231–2.

    Article  CAS  PubMed  Google Scholar 

  21. Park Y-S, Lee S-H, Song SJ, Jun JH, Koong MK, Seo JT. Influence of motility on the outcome of in vitro fertilization/intracytoplasmic sperm injection with fresh vs. frozen testicular sperm from men with obstructive azoospermia. Fertil Steril. 2003;80:526–30.

    Article  PubMed  Google Scholar 

  22. Stalf T, Mehnert C, Hajimohammad A, Manolopoulos K, Shen Y, Schuppe H-C, et al. Influence of motility and vitality in intracytoplasmic sperm injection with ejaculated and testicular sperm. Andrologia. 2005;37:125–30.

    Article  CAS  PubMed  Google Scholar 

  23. Levran D, Ginath S, Farhi J, Nahum H, Glezerman M, Weissman A. Timing of testicular sperm retrieval procedures and in vitro fertilization-intracytoplasmic sperm injection outcome. Fertil Steril. 2001;76:380–3.

    Article  CAS  PubMed  Google Scholar 

  24. Karacan M, Alwaeely F, Erkan S, Çebi Z, Berberoğlugil M, Batukan M, et al. Outcome of intracytoplasmic sperm injection cycles with fresh testicular spermatozoa obtained on the day of or the day before oocyte collection and with cryopreserved testicular sperm in patients with azoospermia. Fertil Steril. 2013;100:975–80.

    Article  PubMed  Google Scholar 

  25. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod. 2003;18:1023–8.

    Article  PubMed  Google Scholar 

  26. Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SEM. Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril. 2004;82:1443–5.

    Article  PubMed  Google Scholar 

  27. Verheyen G, Nagy Z, Joris H, Croo ID, Tournaye H, Steirteghem AV. Quality of frozen-thawed testicular sperm and its preclinical use for intracytoplasmic sperm injection into in vitro-matured germinal-vesicle stage oocytes. Fertil Steril. 1997;67:74–80.

    Article  CAS  PubMed  Google Scholar 

  28. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 5th ed. New York: Cambridge University Press; 2010.

    Google Scholar 

  29. Bechoua S, Berki-Morin Y, Michel F, Girod S. Outcomes with intracytoplasmic sperm injection of cryopreserved sperm from men with spinal cord injury. Basic Clin Androl. 2013;23:14.

    Article  PubMed Central  PubMed  Google Scholar 

  30. Fauque P, Léandri R, Merlet F, Juillard J-C, Epelboin S, Guibert J, et al. Pregnancy outcome and live birth after IVF and ICSI according to embryo quality. J Assist Reprod Genet. 2007;24:159–65.

    Article  PubMed Central  PubMed  Google Scholar 

  31. Fauque P, Jouannet P, Davy C, Guibert J, Viallon V, Epelboin S, et al. Cumulative results including obstetrical and neonatal outcome of fresh and frozen-thawed cycles in elective single versus double fresh embryo transfers. Fertil Steril. 2010;94:927–35.

    Article  PubMed  Google Scholar 

  32. Ohlander S, Hotaling J, Kirshenbaum E, Niederberger C, Eisenberg ML. Impact of fresh versus cryopreserved testicular sperm upon intracytoplasmic sperm injection pregnancy outcomes in men with azoospermia due to spermatogenic dysfunction: a meta-analysis. Fertil Steril. 2014;101:344–9.

    Article  PubMed  Google Scholar 

  33. Aoki VW, Wilcox AL, Thorp C, Hamilton BD, Carrell DT. Improved in vitro fertilization embryo quality and pregnancy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. frozen biopsy samples. Fertil Steril. 2004;82:1532–5.

    Article  PubMed  Google Scholar 

  34. Wood S, Sephton V, Searle T, Thomas K, Schnauffer K, Troup S, et al. Effect on clinical outcome of the interval between collection of epididymal and testicular spermatozoa and intracytoplasmic sperm injection in obstructive azoospermia. J Androl. 2003;24:67–72.

    PubMed  Google Scholar 

  35. Zhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril. 2014;102:998–1005.

    Article  CAS  PubMed  Google Scholar 

  36. Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989;41:183–97.

    Article  CAS  PubMed  Google Scholar 

  37. 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.

    Article  CAS  PubMed  Google Scholar 

  38. Munné S, Estop AM. Chromosome analysis of human spermatozoa stored in vitro. Hum Reprod. 1993;8:581–6.

    PubMed  Google Scholar 

  39. Matsuura R, Takeuchi T, Yoshida A. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa. Asian J Androl. 2010;12:753–9.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Woldringh GH, Besselink DE, Tillema AHJ, Hendriks JCM, Kremer JAM. Karyotyping, congenital anomalies and follow-up of children after intracytoplasmic sperm injection with non-ejaculated sperm: a systematic review. Hum Reprod Update. 2010;16:12–9.

    Article  CAS  PubMed  Google Scholar 

Download references


The authors thank Lysiane Jonval for her help with the statistical analysis, the technical team of the IVF Center of Dijon for their technical help as well as Philip Bastable for spellchecking.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Patricia Fauque.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LD and PF contributed to conception and design, acquisition, analysis and interpretation of data and were involved in drafting the manuscript. PF, CB, PS, FM were involved in revising it critically for important intellectual content; PF, CB, PS, FM, LD, SB, SG have given final approval of the version to be published and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read approved the final manuscript.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Desch, L., Bruno, C., Herbemont, C. et al. Impact on ICSI outcomes of adding 24 h of in vitro culture before testicular sperm freezing: a retrospective study. Basic Clin. Androl. 25, 6 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


Mots clés