Artificial microcontainers for cryopreservation of solitary spermatozoa
Conference: 23th Annual Meeting of the ESHRE
Human Reproduction. 2007. V. 22, Suppl. 1. P. i154-i155.
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Artificial microcontainers for cryopreservation of solitary spermatozoa
Congress: ESHRE 2007
Type: Scientific poster
Topic: ART, laboratory: cryopreservation of gametes
Authors: D. Isaev, S. Zaletov, V. Zaeva, E. Zakharova, R. Shafei, I. Krivokharchenko; Moscow/RU
MeSH: Cryopreservation [E05.760.156]
Biomedical Technology [J01.897.115]
Keywords: ICSI, Sperm cryopreservation, Oligozoospermia, Cryptozoospermia
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Some cases of male infertility caused by severe oligozoospermia (cryptozoospermia) or teratozoospermia only leave a few
spermatozoa suitable for ICSI fertilization. Isolation of functional gametes from testicular or epididymal aspirates requires
considerable workload in azoospermia, while multiple surgical invasions may entail negative consequences. These problems
can be solved by sperm cryopreservation, but there is a high risk of losing unique genetic material in the course of freezing,
storing, and thawing. Placing small groups of spermatozoa in microcontainers eliminates these risks and makes conventional
cryopreservation protocols applicable.
Use of the cryopreservation technique utilizing empty zonae pellucidae (ZP's) of human or animal oocytes, which was
proposed by J. Cohen in 1997, is limited in common clinical practice due to its potential infection and contamination
implications. Furthermore, preparing ZP's requires individual microsurgical evacuation of oocyte cytoplasm; spermatozoa can
get stuck on the internal ZP surface; while some substances found in ZP's might induce an acrosomal reaction.
2. Methods and Materials
Presented is a technique allowing to obtain an artificial ZP analogue, empty microspheres made of agarose gel and used for
cryopreservation of solitary spermatozoa. Such agarose microspheres are inactive and sterile biologically. We used
microspheres made of 2% agarose gel and measuring approximately 100
mm in diameter.
[The hollow microspheres made of agarose gel] see: [The hollow microspheres made of agarose gel]
Motile spermatozoa left after ICSI procedures performed in 18 patients with severe oligoteratozoospermia, were placed in
described microspheres (1 to 10 in each), in the same manner as in ICSI and using the same microinstruments, in 10% PVP
Microspheres loaded with spermatozoa were then placed in a 1:1 solution, Sperm Preparation Medium (MediCult, 1069/1070)
and Sperm Freezing Medium (MediCult, 10670005/10670010), for 5 min. Afterwards, 1 to 5 injected microspheres were put
into 250 mL plastic straws, and these straws were frozen in liquid nitrogen evaporation for 10 min and then placed in liquid
The straws were thawed at room temperature. Microspheres were washed in five Sperm Preparation Medium drops and
incubated at +37
C for an hour. Spermatozoon viability was assessed by motility recovery, as well as in an eosin supravital
test. There was no medical fertilization of human oocytes in these sperm studies.
In total, 318 motile spermatozoa were frozen in 67 microspheres and 19 straws. Two out of 67 microspheres (3%) containing 7
spermatozoa (2% of the total amount) were lost following thawing. Out of 243 remaining spermatozoa, 311 (78%) recovered
motility after incubation. The eosin supravital test demonstrated that 81% of these spermatozoa (251/311) had preserved
[Human spermatozoon inside of agarose microsphere] see: [Human spermatozoon inside of agarose microsphere]
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A new technique is presented (The EAPO Pat.No007992) allowing to obtain and utilize sterile and biologically safe empty
microspheres for efficient cryopreservation of solitary spermatozoa in IVF-ICSI programmes.
[Eurasian Patent â„–007992 (Russian)] see: [Eurasian Patent â„–007992 (Russian)]
Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human
spermatozoa. Hum Reprod. 1997 May;12(5):994-1001.
Walmsley R, Cohen J, Ferrara-Congedo T, Reing A, Garrisi J. The first births and ongoing pregnancies associated with sperm
cryopreservation within evacuated egg zonae. Hum Reprod. 1998 Dec;13 Suppl 4:61-70.
Schuster TG, Keller LM, Dunn RL, Ohl DA, Smith GD. Ultra-rapid freezing of very low numbers of sperm using cryoloops.
Hum Reprod. 2003 Apr;18(4):788-95.
Just A, Gruber I, Wober M, Lahodny J, Obruca A, Strohmer H. Novel method for the cryopreservation of testicular sperm and
ejaculated spermatozoa from patients with severe oligospermia: a pilot study. Fertil Steril. 2004 Aug;82(2):445-7.
Eurasian Patent №007992 (Russian)
Eurasian Patent №007992 for producing agarose microspheres. Document in Russian.
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Human spermatozoon inside of agarose microsphere
Solitary spermatozoa placed into agarose microsphere by similar to ICSI technique and with the same microtools can be frozen by conventional methods.
The hollow microspheres made of agarose gel
These agarose microspheres that can be used as vehicles for single sperm cryopreservation are biologically inert, sterile, prevent sperm losses during
manipulation and provide good post-thaw survival rate. (DIC. 40x).
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[Show abstract] [Hide abstract] ABSTRACT: BACKGROUND: Cryoprotectant free approach successfully removed the impact of physical and chemical damages in preserving human sperm in a vitrification protocol. There is no any report on this technology in farm animal sperm freezing. OBJECTIVE: The aim of the present study was to find the efficacy of cholesterol-loaded cyclodextrin (CLC; 1 mg/60×106) and sucrose (0.1 and 0.2 M) on freezing of the goat epididymal sperm. METHODS: Caudal epididymides (n=5 pairs) were dissected, incised and incubated in the Tris-BSA solution for 15 min, followed by swim-up at room temperature. Sperm was loaded in 0.25 mL French straws and cooled on nitrogen vapor for 3 min then immersed in liquid nitrogen and remained for 48 h. Then the straws thawed by immersing in 37 C waterbath for 30 sec and analyzed. RESULT: The results showed the impact of freezing on the goat epididymal sperm motility, viability and DNA fragmentation that were improved by incorporation of CLC and sucrose (0.2 M). CONCLUSIONS: In conclusion, the goat epididymal sperm was frozen in a cryoprotectant-free freezing model. CLC and 0.4 M sucrose protected the goat epididymal sperm against freezing-induced damages.
- "Therefore, some authors have attempted to cryopreserve small numbers of spermatozoa using various carriers. Biological and nonbiological carriers have been tried for cryopreservation of low numbers of spermatozoa such as empty zona pellucida (Cohen et al., 1997; Walmsley et al., 1998), mini-straws (Desai et al., 2004), ICSI pipette (Gvakharia & Adamson, 2001; Sohn et al., 2003), Volvox globator algae (Just et al., 2004), cryoloops (Schuster et al., 2003; Desai et al., 2004; Isachenko et al., 2005), copper loops (Isachenko et al., 2004b), alginate beads (Herrler et al., 2006), agarose microspheres (Isaev et al., 2007), open pulled straws (Isachenko et al., 2005), microdroplets (Gil-Salom et al., 2000; Isachenko et al., 2005; Jin et al., 2010), cryotop (Endo et al., 2011), cell sleeper (Endo et al., 2012) and cryolock (Stein et al., 2013). In the present study, small volume of goat epididymal spermatozoa was subjected to rapid freezing in WIP, successfully. "
- "When the initial sample is of very poor quality or with very small number of cells, small cell-number or single cell cryopreservation techniques may become useful. Starting in the late 1990's (Cohen & Garrisi, 1997; Cohen et al., 1997), reports on several single sperm cryopreservation techniques showed up in the scientific literature (Walmsley et al., 1998; Gil-Salom et al., 2000; Gvakharia & Adamson, 2001; Just et al., 2004; Herrler et al., 2006; Isaev et al., 2007; Koscinski et al., 2007; Woods et al., 2010). Using these various techniques, researchers reported a wide range of outcomes and recovery efficiency. "
[Show abstract] [Hide abstract] ABSTRACT: Despite interest in cryopreservation of individual or small number of human spermatozoa, to date, little data is available as regards its effectiveness. We systematically reviewed the outcome after cryopreservation of individual or small numbers of human spermatozoa in patients with severe male factor of infertility. We searched the MEDLINE, EMBASE, Cochrane Systematic Reviews, CENTRAL, Web of Science, Scopus databases for relevant studies up to June of 2008. The search used terms referring to cryopreservation of small amount of sperm. Included studies were limited to human studies with no language restrictions. We identified 30 reports including 9 carriers used for cryopreservation of small quantities/numbers of human spermatozoa (7 non-biological and 2 biological carriers). A wide variety of cryopreservation vehicles were reported. The recovery rate of spermatozoa cryopreserved in a known small number varied widely from 59 to 100%. Fertilization rates were in the range of 18-67%. Frozen-thawed spermatozoa, using this method, were subsequently used for intracytoplasmic sperm injection in only five studies, with few pregnancies reported so far. To date, there remains no consensus as to the ideal carrier for cryopreservation of small number of spermatozoa for clinical purposes. Cryopreservation of individual or small numbers of human spermatozoa may replace the need for repeated surgical sperm retrieval. A controlled multicenter trial with sufficient follow-up would provide valid evidence of the potential benefit of this approach.
- "A summary of published reports addressing freezing spermatozoa (ejaculated or surgically retrieved) in microquantities or small numbers was presented (Tables II and III). The quantity of cryopreserved sperm was noted in 20 reports (12 full studies and 8 abstracts) (Cohen et al., 1997; Walmsley et al., 1998; Montag et al., 1999; Suzuki et al., 1999; Borini et al., 2000; Liu et al., 2000; Quintans et al., 2000; Hsieh et al., 2000a; Fusi et al., 2001; Gvakharia and Adamson, 2001; Bouamama et al., 2003; Cesana et al., 2003; Levi-Setti et al., 2003; Sohn et al., 2003; Just et al., 2004; Desai et al., 2004a, b; Hassa et al., 2006; Isaev et al., 2007; Sereni et al., 2008) (Table II), whereas in the remaining studies, the number of cryopreserved spermatozoa was not known (Table III). Eleven of the studies documented sperm function by fertilization/cleavage data (Cohen et al., 1997; Walmsley et al., 1998; Borini et al., 2000; Gil-Salom et al., 2000; Fusi et al., 2001; Nawroth et al., 2002; Desai et al., 2004a, b; Isachenko et al., 2004b; Koscinski et al., 2007; Sereni et al., 2008). "