Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review

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1 Human Reproduction Update, Vol.15, No.2 pp , 2009 Advanced Access publication on December 24, 2008 doi: /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, Cedar Road, Beachwood, OH 44122, USA. Tel: þ ; Fax: þ ; 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 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 Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 154 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 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

3 Methods for cryopreserving spermatozoa in small numbers 155 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 %. 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). 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; 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).

4 Table I Quality assessment of the included cohort studies according to the Newcastle Ottawa Scale Included cohort studies Selection... Comparability... Outcome Total stars... 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.

5 Methods for cryopreserving spermatozoa in small numbers 157 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 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.

6 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% % NA 65% Montag et al. (1999) Full published NA Laser immobilized ejaculated Human zona 92% NA 84% NA sperm 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 % 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 78% NA microspheres ****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.

7 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 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

8 160 AbdelHafez et al. 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 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 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.

9 Methods for cryopreserving spermatozoa in small numbers 161 They reported a post-thaw recovery rate of % (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 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

10 162 AbdelHafez et al. 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

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