107

Sperm Banking: When, Why, and How?Sajal Gupta, Lucky H. Sekhon, and Ashok Agarwal

Cryopreservation is the collection, freezing, and long-term storage of sperm, and is a highly effective method of protecting male fertility potential. Cryopreservation of semen is a vital procedure which can be employed for a variety of purposes, including donor insemina-tion and the preservation of gametes in patients under-going gonadotoxic treatment. It also may be helpful

to fertile couples who experience difficulty conceiving (Thomson et al. 2009). The increasing success of cancer treatment and concerted efforts to ensure quality of life after successful treatment have placed great emphasis on the need to preserve the reproductive capability of young men.

Many health care professionals agree that the option to bank one’s sperm should be offered systematically to all patients who may benefit, including those at risk for future infertility such as patients about to undergo cytotoxic chemotherapy (Achille et al. 2006; Kliesch et al. 1997a). However, this recommendation has yet to become standard practice. In a 2002 survey, only 10% of American physicians reported offering sperm

Contents

Why and When Do Men Need to Bank Their Sperm? ....................................................................................................... 108Couples........................................................................................................................................................................... 108Patients with Cancer ....................................................................................................................................................... 109

Factors That Prevent Patients from Sperm Banking ........................................................................................................... 110How Does Sperm Banking Work: Techniques of Semen Cryopreservation ....................................................................... 110

Preparation and Preselection .......................................................................................................................................... 111Rapid Freezing ............................................................................................................................................................... 111Slow Freezing................................................................................................................................................................. 111

Advantages and Disadvantages of Cryoprotectants ............................................................................................................ 113Glycerol .......................................................................................................................................................................... 113TEST-Yolk Buffer .......................................................................................................................................................... 114

The Effect of Cryopreservation on Sperm Characteristics ................................................................................................. 114ART Outcomes with Banked Semen Specimens ................................................................................................................ 115

Advantages of ICSI and Use of Cryopreserved Spermatozoa ....................................................................................... 115Challenges and Risks Associated with Cryopreservation .............................................................................................. 116

Conclusion .......................................................................................................................................................................... 116References ........................................................................................................................................................................... 116

S. Gupta, L.H. Sekhon, and A. Agarwal ()Center for Reproductive Medicine and Andrology Laboratory and Reproductive Tissue Bank, Glickman Urological & Kidney Institute, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA e-mail: agarwaa@ccf.org

From: Current Clinical Urology: Male Infertility: Problems and Solutions, Edited by: E.S. Sabanegh,DOI: 10.1007/978-1-60761-193-6_12, Springer Science+Business Media, LLC 2011

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banking routinely (Schover et al. 2002a). Semen cryopreservation may be overlooked due to lack of physician awareness regarding the need for fertility preservation and the effectiveness of this option (Joint Council for Clinical Oncology 1998). Furthermore, physicians may overestimate the limitations of poor baseline sperm quality in their patients, leading them to view cryopreservation as futile (Lee et al. 2006). However, with recent developments in reproductive technology, even men with severely impaired sperm parameters can benefit from cryopreservation as pro-cedures such as intracytoplasmic sperm injection (ICSI) require only a few sperm to achieve fertiliza-tion and pregnancy (Hourvitz et al. 2008; Agarwal and Allamaneni 2005; Abdel-Hafez et al. 2009). Zapzalka et al. conducted a survey, revealing that 74% of oncologists were not aware of recent advances in assisted reproductive technology (ART) (Bonetti et al. 2009). Currently, the use of cryopreservation by urolo-gists and gynecologists in in vitro fertilization (IVF) programs remains limited (Abdel-Hafez et al. 2009). Failure to offer this ignores the only possible repro-ductive option available to certain patients. All males of reproductive age facing a disease or gonadotoxic therapy should consider sperm banking before sper-matogenesis is affected (Bonetti et al. 2009).

Physicians are responsible for providing patients with the education necessary to decide for or against cryopreservation. Oncologists and patients agree that better educational materials would help to facilitate this process (Edge et al. 2006). Information leaflets have been identified as a valuable resource to aid health care professionals in discussing cryopreservation although even this avenue remains underutilized (Achille et al. 2006; Schover et al. 2002a; Bazeos et al. 1999). Huyghe et al. conducted randomized trials that explored the effectiveness of multimedia educational tools (Huyghe et al. 2009). These programs were shown to be success-ful in increasing physician knowledge and alleviating decisional conflict in patients (Huyghe et al. 2009). Presenting cryopreservation as the standard of care and sufficiently educating patients facilitates sperm banking.

Why and When Do Men Need to Bank Their Sperm?

Couples

Sperm cryopreservation may serve as a convenient solution for fertile couples who have difficulties coin-ciding intercourse with ovulation, due to reasons such

as travel or geographical separation. Approximately 12% of couples are unable to conceive after 1 year of unprotected intercourse (Eisenberg et al. 2010). Male factor causes account for 30–40% of these cases (Abdel-Hafez et al. 2009). In the most severe cases of male infertility, couples may opt to undergo intrauterine insemination (IUI) using donor sperm (Abdel-Hafez et al. 2009; Crha et al. 2009). Donor insemination programs require cryopreservation of semen samples as the use of frozen semen allows screening of donors for infectious disease, such as HIV and hepatitis B, prior to insemination (Sherman et al. 1986).

Azoospermia is implicated in 10% of male infertility (Abdel-Hafez et al. 2009). Cryopreservation allows for the long-term storage of sperm retrieved from azoosper-mic patients via testicular sperm extraction (TESE) or percutaneous epididymal sperm aspiration (PESA), negating the need for repeat procedures (Donnelly et al. 2001). While some studies have not demonstrated an association of repeated testicular aspiration with any major testicular complications (Westlander et al. 2001), others report that multiple biopsies may pose the risk of inflammation and hematoma at the biopsy site, which can lead to testicular devascularization and fibrosis (Schlegel and Su 1997).

Men may cryopreserve semen samples before undergoing prostate or testicular surgery as serum testosterone levels have been shown to remain low postoperatively, for at least 1 year (Manning et al. 1998). Cryopreservation of sperm before having a vasectomy allows a patient to store his gametes for future use in the event that circumstances or per-sonal preferences change. For some patients who seek to regain their fertility after vasectomy-induced obstructive azoospermia, the use of ICSI with cryo-preserved sperm, originally retrieved by minimally invasive techniques such as TESE and PESA, is a viable and more practical treatment alternative to microsurgical reconstruction of the male genital tract (Schoysman et al. 1993; Devroey et al. 1995; Craft et al. 1993). In cases where the obstructive interval is long term, the success rates of ICSI have been shown to surpass those of vasectomy reversal (Kolettis et al. 2002).

In cases of ejaculatory dysfunction, IUI using cryopreserved semen samples can allow couples to conceive (Craft et al. 1993). Individuals who work in occupations that involve toxic chemicals, ionization radiation, or biological hazards also should consider banking sperm as these exposures may jeopardize their reproductive potential.

109Sperm Banking: When, Why, and How?

Patients with Cancer

In the United States, approximately 1.3 million patients are diagnosed with cancer annually, with an average 5-year survival rate of 60%, resulting in about 9.8 million cancer survivors (American Cancer Society 2005). Most of the common malignancies seen in the reproductive-age male, including testicular cancer and Hodgkin’s disease (Hourvitz et al. 2008), have total survival rates exceeding 95% for early-stage disease (Sant et al. 2007). Williams et al. demonstrated that the average age among 2,680 subjects with testicu-lar cancer was 29.9 (Williams et al. 2009). As full recovery can be achieved in a majority of these young patients, recent efforts have concentrated on mini-mizing treatment-associated morbidity by preserving patient fertility via the cryopreservation of gametes (Hallak et al. 1999).

The quality of spermatozoa in men diagnosed with cancer is often suboptimal, even prior to the initiation of chemotherapy or radiotherapy (Lass et al. 1998). Pre-existing germ cell defects that lead to cancer also cause defective spermatogenesis (Agarwal and Allamaneni 2005). At diagnosis of testicular cancer or Hodgkin’s disease, 50–70% of patients present with oligozoospermia (Howell and Shalet 2002). Many studies suggest that semen quality is significantly correlated with type of malignancy (Lass et al. 1998; Ragni 2003; Bahadur et al. 2005), whereas others failed to demonstrate this association (Padron et al. 1997; Meseguer et al. 2006; Chung et al. 2004). Testicular tumors may exert damaging local effects that can impair sperm quality. Numerous studies have demonstrated reduced sperm counts and motility in patients with testicular cancer (Hourvitz et al. 2008; Crha et al. 2009; Williams et al. 2009). Hodgkin’s disease may activate cytokine secretion, which con-tributes to oxidative stress and impaired fertility (Colpi et al. 2004; Rueffer et al. 2001). Patients with Hodgkin’s disease have been shown to have semen samples with significantly lower sperm concentra-tion and motility than patients with non-Hodgkin’s lymphoma (Botchan et al. 1997). However, other studies failed to confirm this finding (Crha et al. 2009; Agarwal et al. 1996). The fact that this patient popu-lation may face reproductive challenges even before the onset of cancer treatment further highlights the importance of timely cryopreservation.

The degree to which different modalities of cancer therapy are gonadotoxic depends on the agents used, duration of treatment, cumulative dosages, and the age of the patient (Achille et al. 2006). In general, sper-

matogenesis and semen parameters are expected to return to normal levels in 50% of patients 2 years post-treatment and in 85% of patients 5 years after cessa-tion of treatment (Howell and Shalet 2005). However, between 15 and 30% of patients are permanently affected by gonadotoxic treatment and do not recover their reproductive ability (Palermo et al. 1992). It is impossible to predict which patients will be affected permanently (Schrader et al. 2001).

The gonadotoxic effect of chemotherapy is well established (Achille et al. 2006; Bonetti et al. 2009; Schrader et al. 2001; Kobayashi et al. 2001a). Most patients develop azoospermia 12 weeks after com-mencing chemotherapeutic regimens (Schrader et al. 2001). Chemotherapy targets cells outside the G0 phase of the cell cycle, leading to the destruction of prolifer-ating spermatogonia (Bonetti et al. 2009). The degree to which testicular function is impaired is dose and agent dependent (Kobayashi et al. 2001a). More than 50% of patients receive high doses of chemotherapy (Bonetti et al. 2009). Alkylating agents such as cyclo-phosphamide and ifosfamide or combination therapies such as MOPP (mechlorethamine, oncovin, procar-bazine, and prednisone) confer the greatest risk of infertility when taken as a higher cumulative dose for longer periods of time (Bahadur et al. 2000). Ionizing radiation frequently induces azoospermia (Kobayashi et al. 2001a) in a dose-dependent manner, with higher exposures leading to more severe gonadal dysfunction in men (Bahadur et al. 2000). Combination chemora-diotherapy has a synergistic negative effect on infertil-ity (Colpi et al. 2004). Surgical cancer treatments such as retroperitoneal lymph node dissection can also con-tribute to infertility by causing retrograde ejaculation or anejaculation (Puscheck et al. 2004). It is of crucial importance that newly diagnosed male cancer patient’s bank semen samples at the earliest possible stage and before commencing cancer therapy.

Studies have shown that only 5–10% of patients that bank their semen prior to treatment return for IVF treatment using their cryopreserved specimens (Agarwal et al. 2004). Several possible reasons for this include the recovery of spermatogenesis after ces-sation of chemotherapy, death of the patient, anxiety regarding IVF treatment, financial constraints, no plans for more children, and patient uncertainty regarding a long-term prognosis (Bonetti et al. 2009; Crha et al. 2009). This “underutilization” has brought into ques-tion the promotion of routine pretreatment cryopreser-vation (Audrins et al. 1999). However, despite the fact that relatively few patients may return for treatment, cryopreservation should continue to be offered by

110 Gupta et al.

health care professionals as standard care to all patients at risk for iatrogenic infertility (Hourvitz et al. 2008).

Factors That Prevent Patients from Sperm Banking

Despite the well-established link between antineoplas-tic therapy and infertility, only 18–24% of young men with cancer were reported, by survey, to have banked their semen prior to treatment (Schover et al. 2002a). Surveys show that failure of physicians to provide patients with sufficient information in a timely man-ner is one of the main reasons patients fail to utilize cryopreservation to preserve their fertility (de Vries et al. 2009). This could include situations in which the option was not presented entirely (Schover et al. 2002a), the actual risk of infertility was downplayed by the physician (Achille et al. 2006), or patients who were interested failed to receive counseling and refer-ral to a sperm bank (Schover et al. 2002a). Schover et al. (2002a) reported that although more than 90% of oncologists felt that male patients at risk for infertility should be offered sperm banking, only 52% discussed the option with their patients (de Vries et al. 2009).

Even when properly informed regarding infertility risk and cryopreservation, 42–54% of patients did not use sperm banking (Kliesch et al. 1997a). This may be accounted for by patients’ personal preferences, financial constraints, time interval from diagnosis to treatment, and anxiety regarding the consequences of sperm banking. Of those who chose not to bank semen samples, 15% cited lack of interest in parenthood as their primary reason. For newly diagnosed cancer patients, immediate preoccupation with treatment and concerns about survival often take priority over fam-ily planning, resulting in sperm banking becoming a secondary consideration (Achille et al. 2006). Patients are more likely to be reluctant to sperm bank when the process would delay the initiation of treatment (Achille et al. 2006). Some cancer patients require emergency treatment and do not have sufficient time to bank their semen. Patients with leukemia have a relatively reduced time interval between initial diagnosis and the initiation of gonadotoxic therapy (Williams et al. 2009).

As cancer, itself, may already have a profound finan-cial impact, many patients have concerns regarding the cost of sperm banking and continued long-term storage of specimens (Achille et al. 2006). A survey of patients revealed financial constraints to be a major obstacle for 7% of cancer survivors who chose not to bank sperm (Schover et al. 2002a). Cost may play an even larger

role in younger patients with limited or no income (Achille et al. 2006). Schover et al. demonstrated that physicians may overestimate the number of patients for whom sperm banking is not affordable (Schover et al. 2002). Therefore, health care professionals may fail to counsel patients appropriately based on their percep-tion of patient factors. Patient anxiety may arise from misguided beliefs about the efficacy of cryopreserva-tion and risk of transmitting genetic defects to progeny (Achille et al. 2006). However, many detailed studies have shown no increased risk of congenital defects and malignant tumors in the offspring of oncologic patients (Meirow and Schiff 2005). Providing the patient with accurate information may help restore the patient’s perception of the benefits of sperm banking. Rates of cryopreservation might be further improved by presenting sperm banking as a standard practice to patients and their families.

How Does Sperm Banking Work: Techniques of Semen Cryopreservation

Sperm cryopreservation is routinely performed in ART centers and andrology laboratories. The process involves freezing cells to −196°C, the boiling point of liquid nitrogen – a common agent employed in the freezing and storage of spermatozoa. Before banking, semen samples are prepared to select the highest quality sperm for cryopreservation. Vitrification involves the use of cryopreservation agents that minimize cryodamage by decreasing intracellular water content and preventing intracellular ice crystal formation during the freezing process (Hiemstra et al. 2005). Freezing of specimens is accomplished by either rapid or slow freezing tech-niques. A significant post-thaw decrease in sperm qual-ity has been observed, irrespective of the method used (Nallella et al. 2004; Paras et al. 2008) (Fig. 1). In gen-eral, rapid freezing may cause cell lysis by its dehydrat-ing effects and slow freezing may lead to increased ice crystal formation (Kliesch et al. 1997b). Some studies have suggested that the success of freeze–thaw tech-nique may depend on differential thawing temperatures, as thawing at 37°C after rapid freezing and at 22°C after slow freezing was shown to yield the best results (Verheyen et al. 1993). Optimal cooling rates prevent intracellular ice formation. Sources of sperm include semen, retrograde urine, or testicular tissue obtained surgically via conventional testicular biopsy or micro-surgical testicular sperm retrieval (micro-TESE).

111Sperm Banking: When, Why, and How?

Preparation and Preselection

The swim-up method involves centrifugation of a semen sample into pellets, which are then covered with culture medium. The spermatozoa with bet-ter motion characteristics, percentage motility, and viability are separated out as they swim up into the culture medium and can be selected for cryopreserva-tion (Esteves et al. 2002). It has been used by many laboratories as the main sperm washing technique to separate ejaculated spermatozoa from the seminal environment, eliminating dead spermatozoa along with exfoliated epithelial cells, cellular debris, leuko-cytes, and amorphous material (Berger et al. 1985). In comparison to untreated specimens, swim-up-selected cryopreserved sperm have been shown to exhibit faster velocity and progression, higher percentages of intact acrosomes, increased ability to undergo acrosome reaction (Esteves et al. 2002), and better performance in the sperm penetration assay after thawing (Russell and Rogers 1987).

Magnetic-activated cell sorting (MACS) is another preparation technique that has been shown to select motile, viable, morphologically normal spermatozoa displaying higher cryosurvival rates and subsequent fertilization potential (Said et al. 2005; Grunewald et al. 2001, 2006). MACS uses annexin microbeads to immu-nolabel and remove apoptotic spermatozoa (Said et al. 2008). Annexin is a phospholipid-binding protein with a high affinity for phosphatidylserine (PS) – an early

marker of apoptosis when it is externalized to the outer aspect of the cell membrane. Externalized PS indicates impaired membrane integrity (Glander and Schaller 1999). Studies have shown that nonapoptotic sperm selected by MACS before cryopreservation exhibit significantly higher motility (Said et al. 2005), higher levels of intact mitochondria, and decreased pan-caspase activation which leads to decreased rates of sperm apoptosis after thawing compared with sperm that were not selected by MACS (Grunewald et al. 2006).

Rapid Freezing

The Irvine Scientific (IS) method is a fast and con-venient cryopreservation method, allowing for rapid freezing and long-term storage of sperm. The protocol requires the entire volume of freezing medium to be added at one time followed by immersion of specimens in liquid nitrogen (Nallella et al. 2004; Kobayashi et al. 2001b). Contrary to the idea that gradual acclimatiza-tion to very low temperatures protects sperm functional integrity to a greater extent, several studies have shown the IS method to be superior to slow freezing in terms of better post-thaw sperm motility and cryosurvival (Nallella et al. 2004; Hallak et al. 2000).

Slow Freezing

The Cleveland Clinic Foundation (CCF) method of controlled, slow freezing involves the gradual and

Fig. 1. Computer-assisted semen analyzer for prefreeze analysis and post-thaw analysis of count and motility parameters

112 Gupta et al.

Fig. 2. Cryopreservation protocol: shaker for the mixing of the cryoprotectants

Fig. 3. Short-term storage of semen sample in liquid nitrogen tanks

successive addition of freezing media (Fig. 2), fol-lowed by storing specimens first at −20°C for 8 min, then in nitrogen vapors at −96°C for 2 h, followed by immersion in liquid nitrogen at −196°C (Nallella

et al. 2004; Kobayashi 2001b) (Figs. 3 and 4). An analysis by Nallella et al. demonstrated the CCF method to result in thawed sperm with significantly better kinematics – with superior curvilinear velocity,

113Sperm Banking: When, Why, and How?

straight-line velocity, and average path velocity com-pared with sperm cryopreserved by the IS method (Nallella et al. 2004) (Fig. 1). Slow, staged freezing using automated, computerized methods is thought to be more exact and has been reported to limit cryodamage of low-quality sperm (Ragni et al. 1990). However, automated freezers are time-consuming and expensive, requiring up to five times more liquid nitrogen (Paras et al. 2008).

Vitrification

Nawroth et al. described semen cryopreservation achieved by directly plunging spermatozoa into liq-uid nitrogen (cooling rate ~50,000 K/min or more) (Nawroth et al. 2002). The vitrification technique is advantageous, in that it requires no equipment and is straightforward, quick, and inexpensive. However,

the high concentrations (30–50%) of cryoprotectants used in vitrification compared with slow freezing can result in drastically reduced spermatozoal motility. Several special carrier systems for the vitrification solution such as cryoloop, open pulled straws, open straws, and flexipet-denuding pipettes are widely marketed. Cooling can be achieved using either liquid nitrogen or liquid nitrogen vapor phase (Figs. 3 and 4). There is ongoing investigation on optimizing the protocols and determining the precise balance of cryoprotectants and more precisely adjusting their concentration and ratios for the best outcomes with sperm vitrification.

Advantages and Disadvantages of Cryoprotectants

The freezing and thawing process of cryopreserva-tion has detrimental effects on spermatozoa such as impaired sperm motility and vitality and reduced acrosome integrity (Esteves et al. 1998). Various cryo-protectants and cryopreservation methods are used to protect sperm viability post-thawing (Joint Council for Clinical Oncology 1998). The use of a cryoprotective agent to protect sperm cells during the freezing proc-ess is imperative; however, these substances can cause irreparable damage to the ultrastructural morphology of the sperm.

Glycerol

Sperm cells are highly permeable to glycerol, which is a commonly used cryoprotectant (Hallak et al. 2000). Glycerol is often supplemented with citrate or egg yolk, which act as cryobuffers because they contain macromolecules that do not permeate the cell membranes of the sperm. Glycerol serves as an energy source for spermatozoa and also main-tains osmotic pressure by forming hydrogen bonds with membrane phospholipids and sugars (Yildiz et al. 2007). This increases membrane stability and reduces the overall damage to the membrane (Yildiz et al. 2007).

Egg yolk/glycerol mixture has been found to support superior post-thaw outcomes compared with using glycerol alone (Reed et al. 2009). Recent stud-ies have shown that it is possible to replace hen’s egg yolk with soy lecithin when cryopreserving human sperm without adverse effects on post-thaw motility, morphology, or sperm chromatin decondensation.

Fig. 4. Long-term storage of semen sample in liquid nitrogen tank. The figure shows a metal canister containing boxes filled with cryovials

114 Gupta et al.

TEST-Yolk Buffer

A combination of TES [N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pK 7.5] and Tris[(hydroxymethyl)aminomethane] is combined with fresh egg yolk, dextrose, and penicillin–streptomycin to form the cryobuffer known as TEST(TES and Tris)-yolk buffer (TYB) (Nallella et al. 2004). TYB is the preferred cryoprotectant for normal as well as subnormal semen samples, as opposed to using only the glycerol cryoprotectant.

In a study that compared TYB to proprietary media which are commonly used cryobuffer solu-tions such as Sperm Freezing Medium (Cooper Surgical, Trumbull, CT) and Enhance Sperm Freeze (Conception Technologies, San Diego, CA), the sperm that was cryopreserved in TYB was shown to have the longest longevity (Hallak et al. 2000). Sperm mem-branes may be stabilized by an altered phospholipid: cholesterol ratio imposed by the TYB, which may in turn reduce damage by free radicals. Sperm cryopre-served with TYB containing 15% glycerol has higher post-thaw motility and viability than those sperm that are cryopreserved with glycerol alone ( p = 0.004) and it also promotes a higher recovery of motile sperm, and preserves the normal forms of a larger percentage of sperm ( p = 0.04), leading to improved capacitation and sperm penetration.

The Effect of Cryopreservation on Sperm Characteristics

DNA stability, acrosomal integrity, motility, and viabil-ity are features required by a sperm cell that allow it to independently fertilize an egg (Ozkavukcu et al. 2008). These sperm functions must be present in prefreeze specimen and conserved throughout cryopreservation and the post-thaw period for fertilization to be possible with IUI and IVF. Both viability and motility tend to decrease by the same percentage after cryopreserva-tion in both healthy individuals and testicular cancer patients.

Bonetti et al. found a mean post-thaw recovery rate just under 30% in both cancer patients and healthy patients (Bonetti et al. 2009). However, can-cer patients usually have a lower prefreeze quality sperm than do healthy patients (Williams et al. 2009), so their semen tends to have the lowest post-thaw qualities. Pre-existing defects in germ cells or sper-matogenesis, including a possible history of cryp-torchidism, or contralateral or intraepithelial germ

cell neoplasia are common in patients with cancer and could cause low sperm quality (Williams et al. 2009). Also, local endocrine effects of the tumor, systemic endocrine disturbances, autoimmune effects resulting in antisperm antibodies, and the stressors resulting from illness could also lower sperm quality (Hallak et al. 1999). Sperm cryopreservation protocols need to be optimized to compensate for the decrease in cryopreservation-thawing tolerance of spermatozoa in cancer patients.

The literature reports no correlation between post-thaw semen parameters and the duration of semen stor-age (Edelstein et al. 2008); nor was there a correlation between the male partner’s age and the post-thaw semen profile (Hourvitz et al. 2008). The longest duration of freezing with successful IUI outcome of live births have been reported with utilizing sperm stored for 28 years. The freezing and thawing process rather than the duration of storage appears to negatively impact and causes deterioration in sperm quality. Viability of spermatozoa decreases significantly after freezing and thawing. Many studies have shown that the number of viable sperm in a prefreeze sample is reduced by about 50% by cryopreservation. Crystal ice formation outside the cell affects cell morphology by concentrat-ing the surrounding matrix rapidly, leading to high solute content outside the cells and osmotic damage. Cryoprotectant enters the cell through the plasma mem-brane before freezing and is removed during thawing, a process that can cause osmotic injury and damage to the cell membrane (Hallak et al. 2000). This damage inevi-tably results in lower sperm motility. Cryopreservatives like glycerol enter and exit the cell and can lead to swelling and shrinkage that may damage the majority of organelles and deform membrane structures.

Post-thaw motility is reduced in cryopreserved semen samples and depends on the prefreeze motility of the semen sample (Ozkavukcu et al. 2008). Most studies indicate that viability and motility – the most important sperm parameters that determine inde-pendent fertilization capacity – are reduced by 50% between the prefreeze and post-thaw semen samples. Much (but not all) of the reduced motility is likely a direct result of reduced viability caused by damage to cell membranes of the sperm when they are frozen. Some studies have shown that mitochondria contain defects after cryopreservation, but usually this only occurs when the plasma membrane is also damaged. In addition, reactive oxygen species (ROS) can be formed during both freezing and thawing processes, leading to decreased motility through peroxidation of the plasma lipid membrane. However, seminal plasma

115Sperm Banking: When, Why, and How?

contains innate antioxidants, which is one benefit of using unaltered semen during freezing.

Treatment of semen samples with pentoxifyl-line before freezing has been shown to significantly enhance sperm motility, the amplitude of the lateral displacement of the spermatozoal head, and the abil-ity of spermatozoa to undergo the acrosome reaction (Esteves et al. 1998; Schmidt et al. 2004). The posi-tive effects of pentoxifylline may be attributed to its removal of ROS and increase of intracellular cAMP (Esteves et al. 1998). Pentoxifylline has beneficial effects on spermatozoa prior to cryopreservation and is proposed to improve the fertilizing ability of cryopre-served spermatozoa.

ART Outcomes with Banked Semen Specimens

Cryopreservation of spermatozoa provides a readily available gamete reserve for ART and allows coordi-nation with oocyte retrieval. While post-thaw semen quality is often not good enough for IUI, ICSI allows even the poorest quality sperm to fertilize oocytes. Amazingly, the only male factor that determines successful fertilization by ICSI is the production of a single motile sperm – the outcome is independ-ent of other basic semen parameters, not including DNA integrity (Hallak et al. 1999). If a patient cannot produce a sample, minimally invasive procedures are available to recover samples of sperm from the testis and epididymis.

Success rates of IVF and ICSI treatments using cryopreserved semen currently are almost as high as those using fresh semen (van Casteren et al. 2008). The average success rate of achieving pregnancy using cryopreserved semen is 54% and can range between 33 and 73% (van Casteren et al. 2008) To date, there are limited data regarding the outcome of ART treatment using cryopreserved sperm from male cancer survivors (Tournaye et al. 2004). Another study described 258 patients who cryopreserved their semen before chemo-therapy; only 18 of these returned for treatment, with six pregnancies achieved (Audrins et al. 1999).

Advantages of ICSI and Use of Cryopreserved Spermatozoa

The developments in IVF and ICSI have revolution-ized the treatment of male-factor infertility and have made sperm cryopreservation both cost-effective and

the most successful treatment option for men who have viable sperm (Palermo et al. 1992). ICSI reduces the need for storage of many samples and increases the chances for future reproductive success. This is espe-cially helpful in cases where patients only have the opportunity to preserve one or two specimens before initiating cancer treatment (Hourvitz et al. 2008). In a recent report, the ART outcomes in 118 male cancer survivors undergoing 169 IVF–ICSI cycles were stud-ied in a large series of couples treated with IVF–ICSI using cryopreserved sperm stored before cancer ther-apy. The clinical pregnancy rate was 56.8%, compara-ble to the average pregnancy rate achieved with other male-factor patients. The pregnancy outcome in such cases after conventional IVF, before the use of ICSI, was significantly lower. Fertilization failures with IVF were seen in 11% of the patients, as compared with 0.6% after the introduction of ICSI.

Lass et al. reported on 231 men referred for cryopreservation for malignant diseases (Lass et al. 1998). Of the six couples who returned for infertility treatment after chemotherapy, two couples achieved pregnancy after IUI, one couple after IVF, and two couples with ICSI. Given the superior success rates of ICSI over IUI, ICSI should be performed with cryop-reserved sperm to avoid the risk of failed fertilization and avoid the exhaustion of a limited sperm supply.

Other investigators report significantly higher preg-nancy rates and better results using ICSI compared with IVF or IUI (Agarwal et al. 2004; Schmidt et al. 2004; Revel et al. 2005; Kelleher et al. 2001). Certainly, patients with good post-thaw semen quality and sufficient samples can be treated initially by IUI before attempting ICSI–IVF cycles; however, success rates may be low (Agarwal et al. 2004). Agarwal et al. conducted a study in which the success rate with ICSI with cryopreserved sperm was 37% (Agarwal et al. 2004). A recent study from Copenhagen reported a total of 151 ART cycles (55 IUI cycles, 82 ICSI, and 14 ICSI-frozen embryo replacement), in which the clinical pregnancy rate per cycle was 14.8% after IUI and 38.6% after ICSI.

In the past, semen with poor prefreeze or post-thaw quality produced by testicular cancer patients was not conducive to achieving pregnancy by IUI and resulted in low rates of successful pregnancies (Bonetti et al. 2009). However, with the advent of new ARTs such as ICSI, semen samples with the poorest semen parameters can be used to achieve pregnancy because the male patient is required to produce only a single motile sperm. Even though cancer patients have lower quality sperm and quality is further lowered by

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cryopreservation, ICSI circumvents this issue, making it worthwhile to cryopreserve semen samples with even the worst classical sperm parameters (de Vries et al. 2009). ICSI is an option for utilizing the cryopreserved sperm retrieved with TESE and micro-TESE in patients with obstructive as well as nonobstructive azoospermia (Ishikawa et al. 2009). Even though the overall number of embryos transferred and the pregnancy rate per cycle have been reported to be lower in the nonobstructive group, the pregnancy rate per cycle was not different.

Challenges and Risks Associated with Cryopreservation

There are reported risks of cross-contamination from leakage of cryopreserved samples in liquid nitrogen (Clarke et al. 1999). Regulatory bodies have issued current good tissue practice (CGTP) guidelines to prevent any adverse events resulting from these risks. All facilities offering sperm banking are regulated by the FDA and American Association of Tissue Banks (AATB) guidelines and need to be registered with these bodies. FDA has issued guidelines for human cells, tissues, and cellular- and tissue-based products (HCT/P) establishments, and they should follow the requirements in the CGTP regulations.

Conclusion

Cryopreservation is a state-of-the-art technique for storing sperm for a range of indications in the infertil-ity practice. Cryopreserved sperm can be utilized for IVF or ICSI and for multiple, timed artificial inseminations of partner or donor semen. Sperm also can be surgically harvested and cryopreserved before infertility treatment, such as at varicocele ligation, or vasectomy. Cryopreservation has become increasingly recognized as a therapy to alleviate the reproductive morbidity associated with cancer treatment. Chemotherapy, radiation, or surgical therapy or a combination of these have gonadotoxic effects, leading to impairment of sperm quality resulting in infertility. Fertility preservation options should be discussed at an early stage during treatment planning for cancer. Continuing research on developing fast, simple, and cost-effective protocols for semen cryopreservation is needed. The potential of cryopreservation techniques continues to be explored with exciting research, focusing on areas such as cryoprotectant-free sperm vitrification and refining the cooling and warming protocols to obtain optimal outcomes with vitrification.

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