Techniques for cryopreservation of individual or ... - Cleveland Clinic

Human Reproduction Update, Vol.15, No.2 pp. 153–164, 2009 

Advanced Access publication on December 24, 2008 doi:10.1093/humupd/dmn061 

Techniques for cryopreservation of 

individual or small numbers of human 

spermatozoa: a systematic review 

Faten AbdelHafez 1 , Mohamed Bedaiwy 2,3 , Sherif A. El-Nashar 2 , 

Edmund Sabanegh 4 , and Nina Desai 1,5 

1 Department of Obstetrics and Gynecology, Cleveland Clinic Fertility Centre, Cleveland Clinic Foundation, Cleveland, OH, USA 

2 Department of Obstetrics and Gynecology, Assiut University Hospitals, Assiut, Egypt 3 Case Medical Centre, University Hospitals, 

Case Western Reserve University, Cleveland, Ohio, USA 4 Glickman Urological and Kidney Institute, Cleveland Clinic Foundation, Cleveland, 

OH, USA 

5 

Correspondence address. The Cleveland Clinic Fertility Center, 26900 Cedar Road, Beachwood, OH 44122, USA. Tel: þ1-216-839-2907; 

Fax: þ1-216-839-3180; E-mail: desain@ccf.org 

table of contents 

........................................................................................................................... 

† Introduction 

† Methods 

† Results 

† Discussion 

† Conclusions 

background: 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. 

methods: 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. 

results: 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. 

conclusions: 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. 

Key words: Epidydimal sperm / Testicular sperm / Single-sperm cryopreservation / Zona carrier / Cryoloop 

Introduction 

Couples with male factor infertility represent 30–40% of the infertile 

population. Azoospermia accounts for 10% of all male factor cases 

(Chandley, 1979). The birth of the first baby from intracytoplasmic 

sperm injection (ICSI) in 1992 (Palermo et al., 1992) marked a new era 

in the treatment of azoospermic men, who previously had no chance of 

fathering a biologic child. New fertility treatments, such as ICSI combined 

with new advances in minimally invasive sperm recovery from the testis/ 

epididymis (Schoysman et al., 1993; Devroey et al., 1995; Aboulghar et al., 

1997; Ghazzawi et al., 1998; Nicopoullos et al., 2004), have proven to be 

viable and more practical treatment alternatives, in some patients, to 

microsurgical reconstruction of the male genital tract (Craft et al., 1993; 

Schoysman et al., 1993; Devroey et al., 1995). 

& The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 

For Permissions, please email: journals.permissions@oxfordjournals.org

154 AbdelHafez et al. 

Surgical sperm recovery is often the rate-limiting step in successful 

IVF. With epididymal sperm aspirations, sperm recovery can be 

quite variable (45–97%) (Bladou et al., 1991; Craft et al., 1995; 

Lania et al., 2006). Testicular sperm recovery is even more difficult 

and subject to variation. Sperm recovery rates have been reported 

to be only 36–64% with testicular biopsies (Schlegel et al., 1997; 

Palermo et al., 1999; Prins et al., 1999) and even lower in testicular 

aspirates (Hauser et al., 2006). Newer techniques involving testicular 

microdissection offer improved sperm retrieval rates, even when previous 

conventional testicular biopsy has been unsuccessful. Regardless 

of the techniques used, sperm, if successfully recovered, are often 

weakly motile and present in small numbers, necessitating repeat surgical 

interventions if pregnancy is not achieved. This is costly and can 

entail significant risks to the patient. Multiple testicular biopsies can 

lead to inflammation or hematoma at the biopsy site and also to 

major complications such as testicular devascularization and fibrosis 

(Schlegel and Su, 1997). Serum testosterone levels can remain low 

for up to 1 year post-surgery (Manning et al., 1998). Although a 

study by Westlander et al. (2001) suggested that no major complications 

occur after repeated testicular aspiration, testicular damage 

could not be ruled out (Westlander et al., 2001). Further prospective 

studies are needed to address this possibility. 

Cryopreservation of surgically retrieved spermatozoa is therefore a 

very attractive and necessary strategy when dealing with severe male 

factor infertility. Efficient cryopreservation of small numbers of sperm 

reduces the number of surgical interventions and thus avoids the complications 

and expenses associated with repeated surgeries. Using 

cryopreserved sperm also eliminates the logistical issues associated 

with coordinating the women’s oocyte retrieval, with the urologist 

availability for surgical sperm retrieval from her partner. An indirect 

advantage of performing the sperm retrieval prior to initiation of the 

IVF stimulation cycle is that the risk of no sperm being found on the 

day of oocyte retrieval can be avoided. Given the unique characteristics 

of epididymal and testicular spermatozoa, conventional 

methods of sperm cryopreservation may not be optimal. Novel cryopreservation 

approaches are needed to improve recovery and postthaw 

parameters in these highly compromised sperm specimens. 

We performed a systematic review of the literature to summarize 

the current evidence on the feasibility and efficacy of various 

methods for cryopreservation of surgically retrieved sperm for use 

in conjunction with assisted reproductive technology including techniques 

specifically designed for cryopreservation of small quantities/ 

numbers of spermatozoa. 

Methods 

We report this review according to the standards of the Quality of Reporting 

of Meta-analysis of Randomized Trials (QUOROM) and Observational 

Studies (MOOSE) statements (Stroup et al., 2000). 

Search strategy and identification of studies 

We performed an electronic search on MEDLINE, EMBASE, Cochrane 

Systematic Reviews, CENTRAL, Web of Science, Scopus databases for 

relevant studies from inception through June 2008. No language restrictions 

were applied. The search was limited to humans and used the following 

key words: (cryopreserv* OR freez*) AND (small number OR Low OR 

few OR single) AND (sperm*). Moreover, we did a manual search in the 

abstract books of the American Society of Reproductive Medicine (ASRM) 

and the European Society of Human Reproduction and Embryology 

(ESHRE) meetings during the same time period. We decided beforehand 

to include all studies related to cryopreservation of sperm in small quantities 

and numbers, regardless of the source of spermatozoa (ejaculated 

or surgically retrieved). 

Selection criteria and outcomes of interest 

The inclusion criteria for retrieved studies included published studies on 

the cryopreservation of small amount or number of spermatozoa. The 

primary outcome of the study was the post-thaw recovery rate. Secondary 

outcomes included: post-thaw motility, survival, fertilization, cleavage, 

clinical pregnancy, implantation and/or live birth rates. Recovery rate 

was defined as the number of spermatozoa recovered on post-thaw 

divided by the number of spermatozoa initially frozen 100. It was 

reported only for the studies freezing a known number of spermatozoa. 

Motility rate, unless mentioned otherwise, was defined as the number of 

motile spermatozoa divided by total number of spermatozoa 100. 

Two reviewers (F.A. and M.B.) independently reviewed the identified 

reports. Disagreements in study inclusion were resolved by consensus. 

In addition, both reviewers independently reviewed the bibliographies of 

the retrieved articles and the recent reviews for additional studies. 

Data extraction 

A standardized data extraction sheet was developed. One reviewer (F.A.) 

extracted the individual data from each included report. The baseline 

clinical and demographic characteristics of the included studies were 

extracted. The procedure-specific data including the technique and the 

approach for sperm collection and cryopreservation were recorded. 

Study outcomes included post-thaw recovery, motility and viability rates, 

fertilization and cleavage rates, clinical pregnancy and implantation rates. 

Methodological quality of the included 

studies 

The methodological characteristics of the included studies including 

study design, number of participants and procedure-specific characteristics 

were collected. We developed a quality assessment form using the 

validity criteria proposed by Juni et al. for clinical trials and the 

Newcastle–Ottawa Scale for cohort studies (Hallak et al., 2000; Juni 

et al., 2001; Wells et al., 2007). Quality evaluation was performed by 

two reviewers independently (M.B., F.A.) and any disagreement was 

resolved by consensus. 

Results 

Characteristics of the included studies 

Our initial electronic search identified 212 studies published in full text, 

of which 199 were excluded, whereas the manual search of the ASRM 

and ESHRE conferences books retrieved 10 abstracts from the proceedings 

of the relevant meetings in the field and a further 7 

reports were added from bibliography searches. A total of 30 

reports of non-randomized trials were included in the current 

review (Fig. 1). The frozen–thawed sperm was subsequently used 

clinically via ICSI with subsequent transfer of the resultant embryos 

in only five (16.6%) studies. The methodological quality of the included 

studies was evaluated (Table I). 

A summary of published reports addressing freezing spermatozoa 

(ejaculated or surgically retrieved) in microquantities or small

Methods for cryopreserving spermatozoa in small numbers 155 

Figure 1 Flow chart of the included peer-reviewed published 

studies and abstracts presented in the ASRM and ESHRE meetings. 

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). 

Only five studies performed embryo transfer (ET) and reported pregnancy 

results (Walmsley et al., 1998; Gil-Salom et al., 2000; Fusi et al., 

2001; Koscinski et al., 2007; Sereni et al., 2008). Nine types of cryopreservation 

carriers were identified (seven non-biological and two biological). 

The different carrier types included: empty zona pellucida (Cohen 

et al., 1997; Walmsley et al., 1998; Montag et al., 1999; Suzuki et al., 

1999; Borini et al., 2000; Hsieh et al., 2000a; Fusi et al., 2001; Cesana 

et al., 2003; Levi-Setti et al., 2003; Hassa et al., 2006); straws, 

mini-straws and open-pulled straws (Desai et al., 1998; Isachenko 

et al., 2005; Koscinski et al., 2007); direct cryopreservation of 

sperm in microdroplets (Gil-Salom et al., 2000; Quintans et al., 2000; 

Bouamama et al., 2003; Isachenko et al., 2005; Sereni et al., 2008); 

ICSI pipette (Gvakharia and Adamson, 2001; Sohn et al., 2003); 

Volvox globator spheres (Just et al., 2004); alginate beads (Herrler 

et al., 2006); 5-mm copper loop (Nawroth et al., 2002; Isachenko 

et al., 2004b, 2005); cryoloop for microquantities (Schuster et al., 

2003) and for small numbers of sperm (Desai et al., 2004b); and 

agarose microspheres (Isaev et al., 2007). 

Overall outcome of small known numbers of 

spermatozoa frozen using different carriers 

Regarding studies that investigated freezing a selected small known 

number of spermatozoa, the overall average recovery rate was 

79.5% with a range of 59–100%. Whereas the motility rates reported 

were distributed in a wide range starting from 0 to 100%. Survival rate 

was assessed in only three studies, and the overall average survival rate 

was 46.5% with a range of (8–85%). In studies that took a further step 

and performed fertilization, the overall average fertilization rate was 

42.5% with a range of 18–67% (Table II). 

Overall outcome of microquantities of 

spermatozoa frozen using different carriers 

For studies where spermatozoa were frozen in microquantities, the 

overall average survival rate assessed using viability stains was 42% 

with a range of 24–60%. In addition, the post-thaw average number 

of motile spermatozoa was 32.5% with a range of 5–60% from the 

initially frozen spermatozoa. When fertilization was attempted, the 

average fertilization rate was 64.5% with a range of 54–75% (Table III). 

Outcome of IVF cycles using frozen–thawed 

sperm cryopreserved in small numbers 

In this group, only three studies used the frozen–thawed spermatozoa for 

ICSI and ET of the resultant embryos (Walmsley et al., 1998; Fusi et al., 

2001; Sereni et al., 2008). Two studies used the empty zona pellucida 

as the sperm carrier (Walmsley et al., 1998; Fusi et al., 2001), whereas 

Serini et al. froze the spermatozoa in microdroplets on a plastic dish 

(Sereni et al., 2008). In five patients using their frozen–thawed spermatozoa, 

Walmesly et al. reported an average fertilization and cleavage rates of 

65 and 90%, respectively. Thirty-one of the transferred embryos 

implanted resulted in the live birth of two set of twins (Walmsley et al., 

1998). Similarly, Fusi et al. (2001) used thawed spermatozoa frozen in 

empty human zona pellucida for two couples in four cycles. Fifty-seven 

percent of the injected oocytes fertilized, of which 76% cleaved. ET was 

performed in all four cycles with the mean of 3.25 embryos/cycle, resulting 

in the occurrence of one singleton pregnancy. In addition, the study by 

Sereni et al. reported ICSI results using frozen–thawed testicular spermatozoa 

in six cases. The average fertilization and cleavage rates reported 

were 17.6 and 77.8%, respectively. Seventy-one percent of those 

embryos were of good quality. ET in three cases resulted in only one biochemical 

pregnancy (Table II). 

Outcome of IVF cycles using frozen–thawed 

sperm cryopreserved in microquantities 

Regarding sperm freezing in microquantities, only two studies used the 

frozen–thawed spermatozoa for subsequent ICSI and ET (Gil-Salom 

et al., 2000; Koscinski et al., 2007).

Table I Quality assessment of the included cohort studies according to the Newcastle–Ottawa Scale 

Included cohort studies Selection 

................................................ 

Comparability 

................................................................ 

Outcome Total stars 

........................................................... 

........................................................................................................................................................................................................................................................ 

a 

External validity Selection of (a) (b) Adequacy Selection Comparability Outcome 

(intervention) non-exposed Etiology of male Other factors including of follow-up 

cohort 

cohort 

factor infertility age, female factor 

Bouamama et al. (2003) C E Yes No X ** * 

Borini et al. (2000) A F Yes No X *** * * 

Cesana et al. (2003) B E No No X *** * * 

Cohen et al. (1997) B F No No X *** * 

Desai et al. (1998) B E No No X *** * 

Desai et al. (2004b) B E No No X *** * 

(Desai et al. 2004a) A E No No X *** * 

Fusi et al. (2001) A E No No U *** ** 

Gil-Salom et al. (2000) A F No Yes U *** * ** 

Gvakharia and Adamson (2001) B E No No X *** * 

Hassa et al. (2006) B F No No X *** * 

Herrler et al. (2006) C E Yes No X ** * ** 

Hsieh et al. (2000a) A E No Yes X *** * * 

Isachenko et al. (2004b) C E Yes No X ** * * 

(Isachenko et al. 2004a) C E Yes No X ** * * 

Isachenko et al. (2005) B E Yes No X *** * * 

Isaev et al. (2007) B E No No X *** * 

Just et al. (2004) B E No No X *** * 

Koscinski et al. (2007) B F No No U *** ** 

Levi-Setti et al. (2003) D E No No X ** * 

Liu et al. (2000) A E No No X ** * 

Montag et al. (1999) D E No No X ** * 

Nawroth et al. (2002) C E Yes No X ** * * 

Quintans et al. (2000) D E No No X ** * 

Quintans et al. (2000) C E Yes No X ** * * 

156 AbdelHafez et al.


Schuster et al. (2003) C E Yes No X ** * * 

Sereni et al. (2008) A E No No U *** ** 

Sohn et al. (2003) B E Yes No X *** * ** 

Suzuki et al. (1999) D E No No X ** * 

Walmsley et al. (1998) A E No No U *** ** 

External validity: A, truly representative of the azoospermic samples; B, somewhat representative of the average patient seen with the problem of interest; C, selected group; D, no description of the derivation of the cohort. 

Selection of non-exposed cohort: E, drawn from the same source as the intervention cohort (concurrent controls); F, drawn from a different source (historical controls); G, no description of the derivation of the non-exposed cohort. 

Ascertainment of the intervention: categories investigated: H, secure record (e.g. surgical records); J, structured interview; K, written self-report; L, no description. Note: all studies had written self-reports (K). 

Incident disease: Demonstration that outcome of interest was not present at the start of the study: M, yes; N, no. Note: All studies were category M. 

Comparability of cohorts on the basis of the design or analysis: (a) study controls for the most important factor (sperm cryopreservation method); and/or (b) study controls for any additional factor (age, female factors, etc). 

Assesment of outcome: Categories investigated—O, independent blind assessment; P, record linkage; Q, self-report; R, no description. Note: All studies had self-reports (Q). 

Length of follow-up: Question asked: was follow-up long enough for outcomes to occur? S, yes; T, no. Note: In all studies, follow-up was long enough. 

Adequacy of follow-up: U, complete follow-up; all subjects were accounted for. V, subjects lost to follow-up were unlikely to introduce bias because small numbers were lost; .80% had follow-up, or description was provided of those lost. W, 

follow-up rate ,80%, and there was no description of those lost. X, no statement. 

a 

Total stars: a study can be awarded a maximum of one star for each item within the Selection and Outcome categories and a maximum of two stars can be given for comparability. Thus, a perfect study will be awarded a total of nine stars 

distributed as follows: a total of four stars for the selection (a star for each item), two stars for the comparability (a star for each item) and three stars for the outcome (a star for each item). 

The freezing carriers used in those studies were microdroplets on 

ice ‘pills’ (Gil-Salom et al., 2000) and straws (Koscinski et al., 2007). 

In 234 ICSI cycles using frozen–thawed testicular spermatozoa, Gil 

Salom et al. reported an average fertilization and cleavage rates of 

63.2 and 84.6%, respectively. Transferring the resultant embryos in 

211 ET cycles resulted in a 27.8% clinical pregnancy rate per ET, a 

13.1% implantation rate per embryos transferred and 55 live births 

(Gil-Salom et al., 2000). The study by Koscinski et al. performed 

ICSI using frozen–thawed spermatozoa in five cases, resulting in a fertilization 

rate of 54.7% (Koscinski et al., 2007) (Table III). 

Discussion 

Given the unique characteristics of epididymal and testicular spermatozoa, 

conventional methods of sperm cryopreservation are not 

optimal for cryopreservation of small numbers of sperm. Recognition 

of those problems has led to the exploration of methodologies specifically 

designed for cryopreserving limited numbers of motile sperm in 

miniscule volumes to facilitate recovery. The ideal carrier for cryopreservation 

would allow the freezing of multiple tiny aliquots of sperm or 

even individually isolated sperm in small numbers (i.e. 5–10 sperm per 

carrier), thus conserving the very small supply of motile sperm in 

azoospermic patients. This would prevent repeated freezing/thawing 

of the original specimen, and yet allow multiple IVF attempts. 

A number of alternative cryopreservation carriers and techniques 

have been explored to derive a viable clinical solution for this 

problem (Table IV). This approach is necessary if we are to successfully 

treat the severely azoospermic male and not put him at risk of 

medical complications through multiple surgeries for sperm extraction. 

The current work examines techniques that have been applied 

towards this problem. 

Empty zona pellucida as sperm storage 

vessels 

The concept of single-sperm cryopreservation was first introduced by 

Cohen et al. (1997). Their unique and novel idea of using an empty 

zona as a ‘pocket book’ to store individually selected spermatozoa 

has been the most widely used methodology for single-sperm freezing. 

This cryopreservation technique first requires the acquisition of animal 

or human zonae. Micromanipulation techniques and tools are used to 

evacuate cytoplasmic contents of animal or human oocytes. Individually 

selected sperm are then deposited into the empty zonae using an 

ICSI pipette (Cohen et al., 1997; Borini et al., 2000). For cryopreservation, 

the zonae are equilibrated with a test yolk: glycerol cryoprotectant 

agent (CPA) prior to loading into conventional straws. Zonae are 

either vapor-frozen and then plunged in liquid nitrogen or frozen using 

a slow-rate-programmed freezer (Hassa et al., 2006). Empty zonae 

can also be pre-filled with CPA before sperm loading (Levi-Setti 

et al., 2003). Empty oocyte or embryonic zonae from rodents 

(mouse or hamster) (Cohen et al., 1997; Walmsley et al., 1998; 

Montag et al., 1999; Suzuki et al., 1999; Liu et al., 2000; Hsieh et al., 

2000b) as well as from humans (Cohen et al., 1997; Walmsley 

et al., 1998; Borini et al., 2000; Hsieh et al., 2000a; Fusi et al., 2001; 

Cesana et al., 2003; Levi-Setti et al., 2003; Hassa et al., 2006) have 

been successfully used.

Table II Cryopreservation of small known numbers of sperm 

Author Study type Number of Source of sperm Carrier Recovery rate Motility Survival Fertilization 

patients 

rate rate rate 

.......................................................................................................................................................................................................................................................... 

Cohen et al. (1997) Full published 6 Poor ejaculate Human, mouse and hamster zona 73% 82% NA 50% 

*Walmsley et al. (1998) Full published 34 Poor ejaculate, surgically retrieved Human and hamster zonae 75% 67–100% NA 65% 

Montag et al. (1999) Full published NA Laser immobilized ejaculated 

sperm 

Human zona 92% NA 84% NA 

Suzuki et al. (1999) Abstract NA NA Hamster zona pellucida 63–70% 54–73% 65–85% NA 

Borini et al. (2000) Full published 4 Testicular Human zonae 88% 27% NA 23% 

Hsieh et al. (2000a) Full published 16 Ejaculate, surgically retrieved Human and mouse zonae 82% 83% NA NA 

Liu et al. (2000) Full published 9 Testicular Mouse zonae 100% 58% 77% NA 

Quintans et al. (2000) Abstract NA NA Droplets on plastic dish 90–100% NA NA NA 

**Fusi et al. (2001) Abstract 2 Epidydimal Human zona pellucida NA NA NA 57% 

Gvakharia and Adamson (2001) Abstract 18 Ejaculate and testicular ICSI pipetts 92% 52% NA NA 

Bouamama et al. (2003) Full published NA Normal ejaculate 0.5 ml microdrop 100% ,50% NA NA 

Levi-Setti et al. (2003) Full published 10 Ejaculate Human zona 59% 73% NA NA 

Cesana et al. (2003) Abstract NA Poor ejaculate Human zona 80–82% 0–***21% NA NA 

Sohn et al. (2003) Abstract 10 Poor ejaculate ICSI pipetts 80% NA 8% NA 

Desai et al. (2004a) Abstract 4 Epidydimal/testicular Cryoloops 72% NA NA 58% 

Desai et al. (2004b) Full published 10 Poor ejaculate Cryoloop 68% 73% NA 67% 

Just et al. (2004) Full published 15 Poor ejaculate Volvox globator algae 100% 63% NA NA 

Hassa et al. (2006) Full Published NA Normal and poor ejaculate Human zona NA 0–100% NA NA 

Isaev et al. (2007) Abstract 18 Poor ejaculate Agarose gel microspheres 70% of 

microspheres 

78% NA 

****Sereni et al. (2008) Full Published 15 Testicular 5 ml drops in a plastic dish 100% 2% NA 18% 

*First live birth using hamster/human zona for single-sperm freezing. 

**Seventy-six percent of fertilized oocytes cleaved. After ET to two patients in four cycles, one singleton pregnancy resulted. 

***Induced motility by Pentoxifylline. 

****Seventy-eight percent of fertilized oocytes cleaved and a biochemical pregnancy resulted from transfer of six embryos to three patients. 

158 AbdelHafez et al.

Table III Cryopreservation of small quantities of sperm 

Author Study type Number of samples Source of sperm Carrier Survival rate Motility rate Fertilization rate 

.......................................................................................................................................................................................................................................................... 

Desai et al. (1998) Abstract 20 Poor ejaculate Mini-straws NA *40% NA 

**Gil-Salom et al. (2000) Full published 234 ICSI cycle Testicular 100 ml droplets (pills) in cryovials NA NA 63% 

Nawroth et al. (2002) Full published 30 Normal ejaculate 5 mm copper loop 60% *57% 3/4 

Schuster et al. (2003) Full published NA Normal ejaculate Cryoloop NA 45% NA 

Isachenko et al. (2004a) Full published 18 Normal ejaculate 5 mm copper loop NA *52% NA 

Isachenko et al. (2004b) Full published 38 Normal ejaculate 5 mm copper loop NA *50–60% 79% (2PN þ 3PN) 

Isachenko et al. (2005) Full published 16 Poor ejaculate Straw NA *5–10% NA 

Open-pulled straw NA *5–10% NA 

Droplets NA *5–10% NA 

5 mm copper loop NA *,5% NA 

Quintans et al. (2000) Abstract 20 Normal ejaculate Pellets in cryotube 24% NA NA 

Herrler et al. (2006) Full published 15 Normal ejaculate Alginic acid drops 40–50% 20–30% NA 

***Koscinski et al. (2007) Full published 5 Poor ejaculate 30–50 ml instraws NA NA 54% 

*Percentage of post-thaw motility 100 divided by percentage of motility before cryopreservation. 

**Clinical pregnancy and implantation rates were 28 and 13%, respectively. 

***Clinical pregnancy rate was 20%. 

Methods for cryopreserving spermatozoa in small numbers 159


Table IV Summary of different used/suggested carriers 

for cryopreservation of surgically retrieved sperm in 

small quantities/numbers 

Non-biological Biological 

........................................................................................ 

Straws/mini-straws/open-pulled straws Empty zonae of different 

species 

5 mm copper loop Volvox globator algae 

Cryoloops 

Calcium alginate beads 

ICSI pipette 

Microdroplets on dry ice or plastic dish 

Agarose gel microspheres 

Variations in this protocol include making the slit with the aid of a 

LASER (Montag et al., 1999) and using different micropipette sizes 

ranging from 4–15 mm to drill and evacuate the cytoplasmic contents. 

Drilling small holes (7.5 mm) often resulted in oocyte rupture during 

cytoplasmic evacuation. This was not ideal since it increased the risk 

of leaving some host DNA fragments behind. These fragments could 

conceivably adhere to the spermatozoa being cryopreserved and 

later be inadvertently transferred into the oocyte during ICSI. Transfer 

of foreign DNA with sperm as vectors has been reported previously 

(Camaioni et al., 1992; Spadafora 1998). Using larger holes for the 

evacuation of cytoplasmic contents was also problematic, resulting in 

sperm loss. Attempts to seal the hole using oil droplets had limited 

effectiveness and interfered with sperm recovery post-thaw (Montag 

et al. 1999; Levi-Setti et al., 2003). 

Empty zonae as vessels for sperm storage have several positive features. 

This technique is especially valuable in extreme cases of 

oligozoospermia or azoospermia, which might require hours of microscopic 

screening to locate motile sperm. The zonae can be handled 

and visualized microscopically. Cryoprotectants can easily be added 

and removed without loss or dilution of sperm enclosed within the 

zona. Recovery of motile sperm sequestered in the zonae is quite 

simple upon thawing. Walmsley et al. reported the first live births 

using human and hamster zonae for cryopreservation of epidydimal 

and testicular spermatozoa (Walmsley et al., 1998). They performed 

five thaw cycles for five severe oligozoospermic/azoospermic patients. 

Three cycles resulted in pregnancies and two of them ended in the 

birth of two sets of twins. This was followed 3 years later, by a singleton 

pregnancy using oocyte-free human zonae for single epidydimal 

sperm cryopreservation (Fusi et al., 2001). 

Yet, despite its successful application, the routine use of the empty 

zona as a carrier vessel for single-sperm freezing has some drawbacks. 

The most serious limitation is the necessity of using a biologic carrier. 

New FDA and European Tissue Directive regulations, regarding 

exposure of human gametes and embryos to animal products, make 

the use of rodent zonae less feasible. Human zonae availability is 

also very restricted. Moreover, studies suggest lower recovery and fertilization 

rates with human sperm cryopreserved in human zonae, 

possibly as a result of sperm binding to ZP3 and induction of the acrosome 

reaction (Cohen et al., 1997). Furthermore, the process of zona 

preparation is labor-intensive. The technique and hole size can affect 

the retention of motile sperm (Levi-Setti et al., 2003). 

Use of straws for cryopreservation 

of microquantities of sperm 

For surgically retrieved specimens from the epididymis and testis, 

cryopreserving multiple tiny aliquots, each sufficient for a single IVF 

attempt, is highly desirable. In our laboratory, mini-straws have been 

ideal for this task (Desai et al., 1998). Applied to specimens where 

at least one motile sperm was observed per high-powered field (300 

magnification), mini-straws have allowed us to efficiently freeze and 

conserve small aliquots of sperm. This technique utilizes embryo 

cryopreservation straws (0.2-mm diameter) aseptically cut into 

2.5-cm sections. One end is sealed with a plastic plug. A 15–20-ml 

aliquot of the sperm:CPA mixture is carefully loaded into the 

mini-straw using a drawn-out micropipette and the straw is sealed 

with another plastic plug. Five to six mini-straws are placed in conventional 

1.8-ml cryovials. The cryovial containing mini-straws is held in 

the vapor phase for 30 min before plunging into liquid nitrogen. For 

ICSI, one mini-straw can be thawed at a time, without affecting the 

remaining aliquots. Sperm motility and fertilization capacity were not 

affected by cryopreservation in smaller aliquots (unpublished data). 

However, any further reduction in sample volume was not feasible 

using this technique. 

Isachenko et al. used conventional and open-pulled straws for 

vitrifying very small aliquots of 1 or 5 ml of sperm suspension from 

compromised semen samples. To create a closed sterile system, 

they placed the straw containing sperm into another 90-mm straw 

(Isachenko et al., 2005). More recently, Koscinski et al. (2007) used 

straws to freeze 30–50 ml of ejaculated sperm in cryptozoospermic 

patients. Using the straw as a carrier for freezing microquantities of 

sperm is simple and easily applicable. Unfortunately, it is not ideal 

for severely impaired specimens where only small numbers of 

sperm are present, and sperm can be lost due to adherence to the 

vessel wall. Moreover, individually selected sperm cannot be easily 

sequestered within the straw. 

Direct cryopreservation of sperm 

in microdroplets 

To circumvent sperm loss through adherence to the carrier vessel and 

to enable freezing of small volumes, investigators have explored direct 

cryopreservation of sperm in microdroplets (Gil-Salom et al., 2000; 

Quintans et al., 2000). Aliquots of 50 or 100 ml of sperm:CPA 

mixture are placed on the surface of dry ice or cold stainless steel 

plate and then directly plunged into liquid nitrogen. These sperm 

aliquots or ‘pills’ can then be thawed as needed. Gil-Salom et al. 

successfully applied this methodology to testicular sperm (Gil-Salom 

et al., 2000). The clinical pregnancy and implantation rates with ICSI 

using cryopreserved/thawed testicular sperm were comparable with 

those using fresh testicular specimens. Similarly, Isachenko et al. 

vitrified spermatozoa in 40-ml droplets placed directly on dry ice 

but noted that upon warming, the motility was 40% lower than 

fresh control samples (Isachenko et al., 2005). 

Microdroplets have also been used to cryopreserve individually 

selected spermatozoa (Quintans et al., 2000; Bouamama et al., 

2003; Sereni et al., 2008). Quintans et al. (2000) cryopreserved 

selected 4–6 spermatozoa in microdroplets under oil overlay in a 

plastic tissue culture dish. The dish was covered, wrapped with 

plastic stretch film and stored horizontally in a liquid nitrogen tank.


They reported a post-thaw recovery rate of 90–100% (Quintans 

et al., 2000). 

Similarly, Bouamama et al. (2003) isolated up to 100 testicular 

sperm with the aid of an ICSI pipette and deposited them in a 

0.5-ml microdroplet of cryoprotectant medium under an oil overlay. 

The dish containing the microdroplet was then closed, sealed with 

parafilm and held in the vapor phase for 2 h before immersing in 

liquid nitrogen. Post-thaw recovery and motility rates were 100 and 

50%, respectively, but sperm function was not further assessed. 

Recently, Sereni et al. (2008) used a similar microdroplet technique 

to freeze individually selected testicular spermatozoa, obtained from a 

testicular needle aspiration. They applied this technique clinically in six 

cases. Post-thaw recovery rate was 100% (431/431). The pre-freeze 

motility rate was only 3.5%, and on thawing, 67% of sperm retained 

their motility. Nine out of 51 oocytes injected were fertilized. Three 

cases had an ET resulting in one biochemical pregnancy (Sereni 

et al., 2008). 

Neither of the microdroplet techniques for sperm cryopreservation 

has been widely adopted. Direct application of sperm to the surface of 

dry ice with no container physically isolating the specimen is concerning 

in a clinical environment and potentially increases the risk of crosscontamination. 

Culture dishes with microdroplets are also problematic 

since their size and shape make them more difficult to handle and 

store in conventional freezers and liquid nitrogen tanks. Moreover, 

polystyrene dishes do not stand up well to long-term storage in 

liquid nitrogen and cannot be sealed to create a closed system. 

ICSI pipette for single-sperm freezing 

The use of the ICSI pipette as a sterile carrier for individually frozen 

spermatozoa has also been suggested (Gvakharia and Adamson, 

2001; Sohn et al., 2003). Gvakharia and Adamson (2001) cryopreserved 

ejaculated and testicular spermatozoa in ICSI pipettes 

(5–50 spermatozoa in each pipette). The post-thaw recovery and 

motility rates were 92 and 52%, respectively. Sohn et al. compared 

single-sperm freezing in ICSI pipettes using slow and ultrarapid freezing 

methodologies. Twenty-two spermatozoa from severe oligozoospermic 

patients were loaded into each ICSI pipette, which was either 

directly plunged in liquid nitrogen or vapor-frozen for 20 min prior 

to immersion in liquid nitrogen. The recovery and viability rates 

were 90 and 29% for slow freezing and 80 and 8% for ultrarapid freezing, 

respectively (Sohn et al., 2003). 

The use of the ICSI pipette as a vehicle for single-sperm freezing is 

simple and convenient but not practical for long-term storage. The 

ICSI pipette tip is very fragile and can be broken easily. Moreover, 

sperm is being exposed and stored directly in liquid nitrogen raising 

the issue of cross-contamination. 

Volvox globator spheres for sperm storage 

and cryopreservation 

The challenge of single-sperm freezing was addressed in a rather 

unique fashion by Just et al. (2004). This group injected ejaculated 

sperm from 15 patients, with less than 100 motile sperm/ml, into 

spheres of Volvox Globator algae. Colonies from this alga consist of 

1500 to 20 000 cells, held tightly in a sphere-like structure. Spheres 

were secured to a holding pipette and injected with motile sperm 

(n ¼ 8) using an ICSI needle. Algae spheres containing sperm were 

placed in CPA prior to loading into straws, which were vapor-frozen 

and then immersed in liquid nitrogen, following a conventional semen 

cryopreservation protocol. After thawing of straws, sperm were 

extracted from algae spheres using an ICSI needle (Just et al., 2004). 

Despite the 100% on thaw sperm recovery and the .50% on postthaw 

motility, this system has several distinct disadvantages. One is 

exposure of the sperm to genetic material from the algae that can 

be potentially introduced into the oocytes during ICSI. However, 

axenic strains or radioactive irradiation inactivation can be used to circumvent 

this problem (Just et al., 2004). Although algae is inexpensive 

and easy to cultivate and handle during sperm loading, this method 

does involve preparation in advance of the procedure and requires 

a constant source of algae spheres growing in culture. Moreover, 

the new FDA and European Tissue Directive regulations make the 

use of algae (non-human tissue) to store human sperm unacceptable 

in a clinical setting. 

Microencapsulation of sperm 

in alginate beads 

Other investigators have explored sperm microencapsulation in alginate 

beads as a means to cryopreserve small numbers of sperm 

(Herrler et al., 2006). Alginate is a non-toxic polysaccharide derived 

from different species of brown seaweed. Chemically, it is composed 

of repeated units of mannuronic and guluronic acid that can be linked 

by exposure to divalent cations as calcium, resulting in a gel form 

status. Cells and tissues are added to the alginate before gellation. 

Cells can be released from the alginate matrix by calcium chelation. 

In this method, sperm was washed by centrifugation and the pellet 

mixed with CPA. This mixture was immediately mixed with alginic 

acid. The encapsulation was initiated by dropping small aliquots of 

this mixture into a calcium chloride solution. The alginate capsules 

containing sperm were then washed, placed in CPA and frozen in 

straws using a programmable freezer. On thawing, alginate beads 

were dissolved in a sodium citrate solution. Sperm were isolated by 

centrifugation and washed free of alginate (Herrler et al., 2006). 

Good membrane diffusion and the inert nature of the alginate beads 

were the strongest positive features of this microencapsulating 

method. However, the initial testing, using ejaculated sperm specimens, 

revealed an almost 20% reduction in sperm motility. This significant 

decrease in sperm motility with encapsulation was attributed to 

the residual effect of alginic acid on sperm surfaces (Herrler et al., 

2006). This may present a problem when dealing with severely compromised 

samples with poor initial motility. Sperm loss during the 

washing steps to remove alginic acid may also hamper the usefulness 

of this technique for surgically retrieved sperm. 

Cryoloop for sperm cryopreservation 

in microquantities 

The cryoloop has also been explored as a potential means to cryopreserve 

small volumes (Nawroth et al., 2002; Schuster et al., 2003; 

Isachenko et al., 2004b, 2005) and numbers of sperm (Desai et al., 

2004b). The cryoloop was first introduced for embryo cryopreservation 

by Lane et al. (1999). The miniscule fluid volume measuring 

between one-tenth and one-hundredth of a microliter makes this an 

excellent vessel for vitrification protocols that require an ultrarapid 

freezing rate to prevent ice crystallization. Sperm cryopreservation


by vitrification on cryoloops has been explored (Isachenko et al., 

2004a). Sperm motility after vitrification was found to be similar to 

that after conventional slow freeze. However, to date, the conventional 

slow and vapor freeze methods remain to be the commonly used 

sperm cryopreservation techniques. To our knowledge, pregnancies 

using vitrified/warmed sperm have not been reported. 

Schuster et al. (2003) and Desai et al. (2004a, b) combined the 

cryoloop methodology with the conventional slow-freezing protocol 

to cryopreserve very low numbers of sperm in tiny aliquots. The cryoloop 

proved to be a convenient vessel for the storage of tiny aliquots 

of sperm. Sperm loss was minimal since there were no surfaces for 

sperm adherence. Elimination of centrifugation-wash steps also conserved 

sperm (Schuster et al., 2003). 

Desai et al. (2004a, b) proposed the use of the cryoloop as an 

alternative to hamster zona for freezing individual sperm. The individually 

selected ejaculated sperm were picked up using an ICSI pipette 

and deposited directly on a cryoprotectant film covering the nylon 

loop (Desai et al., 2004b). Single-sperm freezing on cryoloops 

proved to be straightforward. No difference was observed in postthaw 

motility after cryopreservation on loops versus in conventional 

vials. Among the individually cryopreserved sperm, 73% retained motility. 

Further sperm-function testing demonstrated that these individually 

cryopreserved sperm were indeed capable of inducing sperm head 

decondensation and pronuclear formation when injected into human 

oocytes (Desai et al., 2004b). 

The cryoloop as a carrier was further tested using individual sperm 

isolated from both epididymal and testicular specimens (Desai et al., 

2004a). Cryoloops were loaded with two to eight individually selected 

epididymal or testicular sperm, vapor-frozen and then immersed in 

liquid nitrogen. Seventy-two percent (23/32) of the individually 

selected and cryopreserved epididymal/testicular sperm were recovered. 

On thaw, motility was usually minimal (slight head motion or 

twitching), but the majority of sperm had flexible tails, an indicator 

of potential viability. Both epididymal and testicular sperm cryopreserved 

on cryoloops were capable of fertilizing oocytes (Desai et al., 

2004a). 

As a carrier, the inert, non-biologic nature of the nylon material of 

the cryoloop helps to circumvent many of the problems associated 

with the methodologies involving animal zonae or algae spheres. 

Sperm can be sequestered easily but without encapsulation, so 

there is no need to expose them to compounds that might decrease 

motility, such as alginic acid. Cryoloops can be commercially purchased 

and require no additional preparation. Cryovials are more 

easily stored in conventional liquid nitrogen storage containers and 

freezers than tissue culture dishes or micropipettes containing 

sperm. The major drawback of the cryoloop as a carrier is that it is 

an open system. Since sperm are directly exposed to liquid nitrogen 

within the vial, the potential risk of cross-contamination has been 

raised. The miniscule volumes of fluid on the loop, generally less 

than one-tenth of a microliter in the case of single-sperm freezing, realistically 

make this risk negligible. However, new FDA and European 

Tissue Directive regulations discourage the use of open systems for 

gamete preservation. Modifications of the original technique by enclosure 

of cryoloop-containing vials (Larman et al., 2006), storage and 

freezing in the vapor phase and sterile liquid nitrogen delivery 

systems are all potential approaches to improving the widespread 

acceptance of cryoloops as carrier vessels for individually selected 

sperm. Future clinical trials with sperm frozen on cryoloops in a 

closed system are needed to further evaluate this carrier system. 

Sperm freezing in agarose microspheres 

Recently, Isaev et al. used 2% agarose microspheres (100 mm in diameter) 

as a non-biological analogue to zona pellucida (Isaev et al., 2007). 

Using micromanipulation tools, each microsphere was loaded with 

1–10 spermatozoa and placed in CPA solution for 5 min to equilibrate. 

One to five agarose microspheres loaded with sperm were 

then put in to 0.25 cc plastic straws, vapor-frozen and then plunged 

into liquid nitrogen. Using this technique, a total of 318 motile 

spermatozoa were cryopreserved in 67 microspheres. On thaw, 

78% of recovered spermatozoa were motile (Isaev et al., 2007). 

Two microspheres containing seven sperm were lost. Agarose microspheres 

represent another inert non-biologic carrier for freezing small 

numbers of sperm. More details on the steps involved in microsphere 

preparation and use, along with sperm function testing data, will 

however be needed to assess functionality as a potential carrier for 

clinical application. 

Conclusions 

Cryopreservation of surgically retrieved spermatozoa from the epididymis 

and testis is a valuable component in effective treatment and 

management of male infertility, reducing the necessity of repeat surgeries. 

Biological and non-biological carriers have been tried for cryopreservation 

of low numbers of spermatozoa. Thawed spermatozoa 

were used for subsequent ICSI and ET in only five studies. No prospective, 

randomized controlled trials were performed to show that 

any single carrier is superior to the others. It is clear that conventional 

methodology cannot effectively address these problems. Surgically 

retrieved specimens are often heavily loaded with red blood cells, 

cellular debris, testicular cells and a large number of dead or immotile 

sperm. Concentrating such samples into small volumes to cryopreserve 

can actually have a detrimental effect on the few observed 

motile sperm (Aitken and Clarkson, 1988; Mortimer and Mortimer, 

1992). Lack of easily implemented technology to handle such cases 

remains a major deterrent to single-sperm freezing. 

More than a decade had passed since pregnancies were described 

after the use of human or hamster zonae for storing one or few spermatozoa. 

To date, there is a limited use of this technology by urologists 

and gynecologists in the majority of the IVF programs. It is 

indeed a missed opportunity to improve on the quality of care provided 

to that subset of patients. Failure of this technology to have 

mainstream use remains multi-factorial. First, there is a paucity of 

clinical data on its effectiveness. In addition, there is a lack of the 

ideal container or vehicle that could be used universally. Even the 

cryoloop that we proposed 3 years ago has its own limitations and 

drawbacks. We are now trying some modifications to the cryoloop 

technique to be more feasible for clinical application. 

Novel cryopreservation technology specifically designed to handle 

small numbers and quantities of sperm needs to be further explored. 

To date, there have been few published studies on cryopreservation of 

small numbers of testicular or epididymal sperm in extremely impaired 

surgical specimens. The current evidence is not sufficient to support 

the use of one technology over the other. Well-designed clinical


trials with appropriate sample sizes are needed to assess the feasibility 

and efficiency of various low sperm count freezing methodologies. 

References 

Aboulghar MA, Mansour RT, Serour GI, Fahmy I, Kamal A, Tawab NA, 

Amin YM. Fertilization and pregnancy rates after intracytoplasmic 

sperm injection using ejaculate semen and surgically retrieved sperm. 

Fertil Steril 1997;68:108–111. 

Aitken RJ, Clarkson JS. Significance of reactive oxygen species and 

antioxidants in defining the efficacy of sperm preparation techniques. 

J Androl 1988;9:367–376. 

Bladou F, Grillo JM, Rossi D, Noizet A, Gamerre M, Erny R, Luciani JM, 

Serment G. Epididymal sperm aspiration in conjunction with in-vitro 

fertilization and embryo transfer in cases of obstructive azoospermia. 

Hum Reprod 1991;6:1284–1287. 

Borini A, Sereni E, Bonu MA, Flamigni C. Freezing a few testicular 

spermatozoa retrieved by TESA. Mol Cell Endocrinol 2000;169:27–32. 

Bouamama N, Briot P, Testart J. Comparison of two methods of 

cryoconservation of sperm when in very small numbers. Gynecol 

Obstet Fertil 2003;31:132–135. 

Camaioni A, Russo MA, Odorisio T, Gandolfi F, Fazio VM, Siracusa G. 

Uptake of exogenous DNA by mammalian spermatozoa: specific 

localization of DNA on sperm heads. J Reprod Fertil 1992;96:203–212. 

Cesana A, Novara P, Bianchi S, Marras A, Albani E, Negri L, Levi-Setti PE. 

Sperm cryopreservation in oligo-asthenospermic patients. The 19th 

Annual Meeting of the ESHRE, Madrid, Spain. Hum Reprod 2003; 

18:xviii76. 

Chandley AC. The chromosomal basis of human infertility. Br Med Bull 

1979;35:181–186. 

Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, 

Scott RT. Cryopreservation of single human spermatozoa. Hum 

Reprod 1997;12:994–1001. 

Craft I, Bennett V, Nicholson N. Fertilising ability of testicular 

spermatozoa. Lancet 1993;342:864. 

Craft I, Tsirigotis M, Bennett V, Taranissi M, Khalifa Y, Hogewind G, 

Nicholson N. Percutaneous epididymal sperm aspiration and 

intracytoplasmic sperm injection in the management of infertility due 

to obstructive azoospermia. Fertil Steril 1995;63:1038–1042. 

Desai N, Glavan D, Goldfarb J. A convenient technique for 

cryopreservation of micro quantities of sperm. Fertil Steril 1998; 

S197–S198. Annual meeting program supplement. 

Desai N, Culler C, Goldfarb J. Cryopreservation of single sperm from 

epidydimal and testicular samples on cryoloops: preliminary case 

report. Fertil Steril 2004a;82:S264. 

Desai NN, Blackmon H, Goldfarb J. Single sperm cryopreservation on 

cryoloops: an alternative to hamster zona for freezing individual 

spermatozoa. Reprod Biomed Online 2004b;9:47–53. 

Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van 

Steirteghem A, Silber S. Pregnancies after testicular sperm extraction 

and intracytoplasmic sperm injection in non-obstructive azoospermia. 

Hum Reprod 1995;10:1457–1460. 

Fusi F, Calzi F, Rabellotti E, Papaleo E, Gonfiantini C, Bonzi V, Santis LD, 

Ferrari A. Fertilizing capability of frozen-thawed spermatozoa recovered 

from microsurgical epidydimal sperm aspiration and cryopreserved in 

oocyte-free human zona pellucida. Hum Reprod 2001;16:117. 

Ghazzawi IM, Sarraf MG, Taher MR, Khalifa FA. Comparison of the 

fertilizing capability of spermatozoa from ejaculates, epididymal 

aspirates and testicular biopsies using intracytoplasmic sperm injection. 

Hum Reprod 1998;13:348–352. 

Gil-Salom M, Romero J, Rubio C, Ruiz A, Remohi J, Pellicer A. 

Intracytoplasmic sperm injection with cryopreserved testicular 

spermatozoa. Mol Cell Endocrinol 2000;169:15–19. 

Gvakharia M, Adamson GD. A method of successful cryopreservation of 

small numbers of human spermatozoa. Fertil Steril 2001;76:S101. 

Hallak J, Sharma RK, Wellstead C, Agarwal A. Cryopreservation of human 

spermatozoa: comparison of TEST-yolk buffer and glycerol. Int J Fertil 

Womens Med 2000;45:38–42. 

Hassa H, Gurer F, Yildirm A, Can C, Sahinturk V, Tekin B. A new 

protection solution for freezing small numbers of sperm inside empty 

zona pellucida: Osmangazi-Turk solution. Cell Preserv Technol 2006; 

4:199–208. 

Hauser R, Yogev L, Paz G, Yavetz H, Azem F, Lessing JB, Botchan A. 

Comparison of efficacy of two techniques for testicular sperm 

retrieval in nonobstructive azoospermia: multifocal testicular sperm 

extraction versus multifocal testicular sperm aspiration. J Androl 2006; 

27:28–33. 

Herrler A, Eisner S, Bach V, Weissenborn U, Beier HM. Cryopreservation 

of spermatozoa in alginic acid capsules. Fertil Steril 2006;85:208–213. 

Hsieh Y, Tsai H, Chang C, Lo H. Cryopreservation of human spermatozoa 

within human or mouse empty zona pellucidae. Fertil Steril 2000a; 

73:694–698. 

Hsieh YY, Tsai HD, Chang CC, Lo HY. Sperm cryopreservation with 

empty human or mouse zona pellucidae. A comparison. J Reprod Med 

2000b;45:383–386. 

Isachenko E, Isachenko V, Katkov II, Rahimi G, Schondorf T, Mallmann P, 

Dessole S, Nawroth F. DNA integrity and motility of human 

spermatozoa after standard slow freezing versus cryoprotectant-free 

vitrification. Hum Reprod 2004a;19:932–939. 

Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, 

Van Der Ven H. Cryoprotectant-free cryopreservation of human 

spermatozoa by vitrification and freezing in vapor: effect on motility, 

DNA integrity, and fertilization ability. Biol Reprod 2004b;71:1167–1173. 

Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, 

Nawroth F, Dessole S, Katkov II, van der Ven H. Clean technique for 

cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed 

Online 2005;10:350–354. 

Isaev DA, Zaletov SY, Zaeva VV, Zakharova EE, Shafei RA, 

Krivokharchenko IS. Artificial microcontainers for cryopreservation of 

solitary spermatozoa. Hum Reprod 2007;22:i154. 

Juni P, Altman DG, Egger M. Systematic reviews in health care: assessing 

the quality of controlled clinical trials. BMJ 2001;323:42–46. 

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;82:445–447. 

Koscinski I, Wittemer C, Lefebvre-Khalil V, Marcelli F, Defossez A, 

Rigot JM. Optimal management of extreme oligozoospermia by an 

appropriate cryopreservation programme. Hum Reprod 2007; 

22:2679–2684. 

Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification 

of mammalian oocytes and embryos. Nat Biotechnol 1999; 

17:1234–1236. 

Lania C, Grasso M, Fortuna F, De Santis L, Fusi F. Open epididymal sperm 

aspiration (OESA): minimally invasive surgical technique for sperm 

retrieval. Arch Esp Urol 2006;59:313–316. 

Larman MG, Sheehan CB, Gardner DK. Vitrification of mouse pronuclear 

oocytes with no direct liquid nitrogen contact. Reprod Biomed Online 

2006;12:66–69. 

Levi-Setti PE, Albani E, Negri L, Cesana A, Novara P, Bianchi S. 

Cryopreservation of a small number of spermatozoa in yolk-filled 

human zonae pellucidae. Arch Ital Urol Androl 2003;75:195–198.


Liu J, Zheng XZ, Baramki TA, Compton G, Yazigi RA, Katz E. 

Cryopreservation of a small number of fresh human testicular 

spermatozoa and testicular spermatozoa cultured in vitro for 3 days 

in an empty zona pellucida. J Androl 2000;21:409–413. 

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. 

Montag M, Rink K, Dieckmann U, Delacretaz G, van der Ven H. 

Laser-assisted cryopreservation of single human spermatozoa in 

cell-free zona pellucida. Andrologia 1999;31:49–53. 

Mortimer D, Mortimer ST. Methods of sperm preparation for assisted 

reproduction. Ann Acad Med Singapore 1992;21:517–524. 

Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, 

Mallmann P, Dattena M, Capobianco G, Peters D et al. Vitrification of 

human spermatozoa without cryoprotectants. Cryo Letters 2002; 

23:93–102. 

Nicopoullos JD, Gilling-Smith C, Almeida PA, Norman-Taylor J, Grace I, 

Ramsay JW. Use of surgical sperm retrieval in azoospermic men: a 

meta-analysis. Fertil Steril 2004;82:691–701. 

Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after 

intracytoplasmic injection of single spermatozoon into an oocyte. 

Lancet 1992;340:17–18. 

Palermo GD, Schlegel PN, Hariprashad JJ, Ergun B, Mielnik A, Zaninovic N, 

Veeck LL, Rosenwaks Z. Fertilization and pregnancy outcome with 

intracytoplasmic sperm injection for azoospermic men. Hum Reprod 

1999;14:741–748. 

Prins GS, Dolgina R, Studney P, Kaplan B, Ross L, Niederberger C. Quality 

of cryopreserved testicular sperm in patients with obstructive and 

non-obstructive azoospermia. J Urol 1999;161:1504–1508. 

Quintans CJ, Donaldson MJ, Asprea I, Geller M, Rocha M, Pasqualini RS. 

Development of a novel approach for cryopreservation of very small 

numbers of spermatozoa. Hum Reprod 2000;15:99. 

Schlegel PN, Su LM. Physiological consequences of testicular sperm 

extraction. Hum Reprod 1997;12:1688–1692. 

Schlegel PN, Palermo GD, Goldstein M, Menendez S, Zaninovic N, 

Veeck LL, Rosenwaks Z. Testicular sperm extraction with 

intracytoplasmic sperm injection for nonobstructive azoospermia. 

Urology 1997;49:435–440. 

Schoysman R, Vanderzwalmen P, Nijs M, Segal L, Segal-Bertin G, Geerts L, 

van Roosendaal E, Schoysman D. Pregnancy after fertilisation with 

human testicular spermatozoa. Lancet 1993;342:1237. 

Schuster TG, Keller LM, Dunn RL, Ohl DA, Smith GD. Ultra-rapid freezing 

of very low numbers of sperm using cryoloops. Hum Reprod 2003; 

18:788–795. 

Sereni E, Bonu MA, Fava L, Sciajno R, Serrao L, Preti S, Distratis V, 

Borini A. Freezing spermatozoa obtained by testicular fine needle 

aspiration: a new technique. Reprod Biomed Online 2008;16:89–95. 

Sohn JO, Jun SH, Park LS, Kim EK, Chung TG, Lee DR. Comparison of 

recovery and viability of sperm in ICSI pipette after ultra rapid 

freezing or slow freezing. Fertil Steril 2003;80:S128. 

Spadafora C. Sperm cells and foreign DNA: a controversial relation. 

Bioessays 1998;20:955–964. 

Stroup DF, Berlin JA, Morton SC, Olkin I, Williamson GD, Rennie D, Moher D, 

Becker BJ, Sipe TA, Thacker SB. Meta-analysis of observational 

studies in epidemiology: a proposal for reporting. Meta-analysis Of 

Observational Studies in Epidemiology (MOOSE) group. JAMA 2000;283: 

2008–2012. 

Suzuki N, Suzuki Y, Sato T, Matsubara H, Ikuta K, Suzumori K. 

Cryopreservation of a few spermatozoa in hamster zonae pellucidae. 

Hum Reprod 1999;14:289. 

Wells GA, Shea A, O’Connell D, Peterson J, Welch J, Losos M et al. The 

Newcastle-Ottawa Scale (NOS) for Assessing the Quality of Nonrandomised 

Studies in Meta-Analyses (Homepage on the Internet). Ottawa Health 

Research Institute: Ottawa, c1996–2006. Available from: http:// 

www.ohri.ca/programs/clinical_epidemiology/oxford.htm [updated 9 

June 2007; cited 6 September 2007]. 

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; 

13(Suppl. 4):61–70. 

Westlander G, Rosenlund B, Soderlund B, Wood M, Bergh C. Sperm 

retrieval, fertilization, and pregnancy outcome in repeated testicular 

sperm aspiration. J Assist Reprod Genet 2001;18:171–177. 

Submitted on January 18, 2008; resubmitted on November 4, 2008; accepted on 

December 2, 2008

Techniques for cryopreservation of individual or ... - Cleveland Clinic

Create successful ePaper yourself

Delete template?

Save as template?