Fig. 14.1
Preparation of the sample for cryopreservation. The sample is placed in an incubator at 37 °C for complete liquefaction before conducting semen analysis [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Within 1 h of specimen collection , an aliquot of freezing medium equal to 25% of the original specimen volume is added to the centrifuge tube with a sterile pipette. The specimen with the freezing media is gently mixed on a test tube rocker for 5 min (Fig. 14.2). The steps of addition of the freezing media and mixing of the specimen are repeated three times or until the volume of freezing media added is equal to the original specimen volume (Fig. 14.3). After addition of TYB, sperm motility is examined in the cryodiluted sample using a fixed cell chamber and phase microscope.
Fig. 14.2
Mixing of the specimen with the freezing media for 5 min on a test tube rocker [liquefy, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Fig. 14.3
Stepwise addition of Test-Yolk buffer to patient sample. Volume of Test-Yolk buffer equal to ¼ volume of patient sample—added four times, or until total volume in test tube has doubled [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
The percent motility is recorded as “ pre-cryo motility %.” Using a sterile serological pipette the well-mixed, cryodiluted semen is equally aliquoted into pre-labelled vials (Fig. 14.4). Exposure to freezing conditions should occur within 1.5 h of specimen collection. Two cryovials are loaded into the cryocanes and placed upright in –20° C for 8 min. Labelled portion of cryocanes should be facing the ground while placing the cryovials upright, with the orange top facing (Fig. 14.5) before submersion in the vapour phase for 2 h (Fig. 14.6). Finally, the cryocanes are flipped and immersed in liquid nitrogen for short-term storage. Once the client has completed sperm banking the samples are transferred from short-term storage to long-term storage (Fig. 14.7). The major drawback of this technique is the formation of ice crystals if the cooling is too fast and cell shrinkage if the cooling is too slow.
Fig. 14.4
Even distribution of cryodiluted patient sample into cryovials using a sterile serological pipette [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Fig. 14.5
Proper placement of cryovials into cryocanes before immersion in LN2 vapours [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Fig. 14.6
Lowering of canes containing cryovials into LN2 tank in vapour phase [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Fig. 14.7
Transfer of samples from short-term to long-term storage [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Rapid Freezing
The aim of utilizing the rapid freezing technique is to minimize the toxicity caused by the cryoprotectant and to lessen the osmotic membrane damage by inhibiting the ice crystal formation [23]. This is achieved by bringing the samples in direct contact with nitrogen vapours at –80 °C after addition of the cryoprotectant. The entire volume of cryoprotectant is added drop by drop at the same time to result in a 1:1 freezing ratio. The mixture is then transferred to a cryovial with a maximum of 1.0 mL volume per vial. Static vapour exposure is performed with the vials placed 3 cm above the liquid nitrogen surface for a 30 min before being submerged in liquid nitrogen [26]. Controlled freezing cannot be achieved in either slow or rapid freezing. This can be overcome by using a programmable freezer where the desired temperature is achieved by a preloaded temperature set-up.
Sperm Vitrification
Sperm vitrification was described earlier in 1949 by Polge in Nature [27]. However, the vitrification protocols still require a lot of work and standardization. Several reports provide different approaches for standardization of the sperm vitrification procedure. The investigators have tried different devices (open or closed), different media concentrations media or exposure time [28]. The relative permeability of cryoprotectants determines the level of osmotic stress to which the spermatozoa are exposed. This is an important factor in sperm cryodamage .
Sperm are cooled at a very fast rate of –1000 °C per minute. The basic principle underlying the vitrification technique is to create high viscosity in the solution and produce a glass-like solidification without the formation of ice crystals. The technique has not been standardized as it is difficult to perform the cooling at such high rates and standardization of the high concentration of cryoprotectants has not been achieved. Cryoprotectant-free vitrification using sucrose is associated with better outcomes of post-thaw sperm motility, plasma membrane integrity and acrosome integrity [29].
The principle for vitrification is that small volumes of the cryopreserved specimen mixture are dropped directly into liquid nitrogen to achieve high rate of cooling [30]. Non-permeable cryoprotectants are used for vitrification. Small volume of sperm suspension is mixed with sperm wash media supplemented with 5% HSA and sucrose. The vial is placed at the bottom of a metal strainer and immersed into the liquid nitrogen. The sperm suspension is then dropped with a micropipette directly into liquid nitrogen. The vials with solid spheres in them are packed and placed in liquid nitrogen [15].
Home Sperm Banking
Home sperm banking is a novel option for men who choose to freeze their specimens but prefer to provide semen samples from the comfort of their home. Banking from home helps avoid emotional stress as well as other privacy-related issues. Patients who may be interested in this service include men who have cancer and are going through gonadotoxic treatments, military personnel before deployment and men undergoing infertility treatment. Semen specimens remain viable through a short transport cycle utilizing the specialized sperm collection and transport system called NextGen® [31, 32]. After receipt of the NextGen kit with the sample, cryopreservation is done as per the in-house protocol based on slow cryopreservation technique [31]. The kit retains adequate sperm viability during transit prior to long-term freezing in the tissue bank. Semen samples from infertile men with and without cancer were collected onsite and offsite and shipped using NextGen. Total motile sperm and motility were comparable and no significant differences were observed in the cryosurvival rates of semen samples collected offsite and transported via the NextGen [31].
Sperm Preparation for Cryopreservation
Many times the sample has to be processed before cryopreservation. This is necessary to eliminate the contaminating round cells, leukocytes, dead cells and debris and seminal plasma to obtain a good-quality sperm before freezing. Both abnormal sperm and leukocytes produce reactive oxygen species causing sperm damage and DNA fragmentation. In abnormal semen samples , preparation of sperm has been used to increase the percentage of motile, morphologically normal sperm. This resulted in increased success rates in reproductive treatment outcome in either IUI or ART [33].
Freezing significantly increases abnormally dead, immotile spermatozoa with low DNA integrity after freeze-thawing [34–39]. Preparation of post-thawed spermatozoa is performed to discard the cryoprotectant and select the best quality spermatozoa before using for ART . Sperm preparation before [38, 40–42] and after cryopreservation has been studied [43–45]. Significant improvement in the number of motile spermatozoa with reduced incidence of apoptosis was reported in sperm prepared before freezing [43–46] compared to after freezing [43–45]. Two common methods used to obtain a highly motile sperm fraction are the separation of sperm by a double-density gradient and swim-up technique. Each method has its own advantages and disadvantages [47].
Sperm Preparation by Double-Density Gradient
In this technique, sperm are separated based on their density. After centrifugation , the morphologically normal, highly motile sperm are collected from the pellet at the bottom of the tube. A colloidal suspension of silica particles stabilized with a covalently bonded hydrophilic silane supplied in HEPES medium is used to prepare the double-density gradient. To avoid the detrimental effects of reactive oxygen species produced by high centrifugation speeds, centrifugation speeds are kept no higher than 300 × g to which are detrimental to the sperm. In brief, after the sample has completely liquefied, a manual semen analysis is done for volume, concentration and motility. Lower phase or high density (90%) gradient and the upper phase or the low density (45%) gradient are used. Both gradients and sperm wash medium (modified HTF with 5.0 mg/mL human albumin) are brought to room temperature before loading the sample.
Using a 15 mL conical centrifuge tube, 2 mL of the lower phase is placed at the bottom and carefully layered with 2 mL of the upper phase. A well-mixed semen sample is placed on the upper phase and the sample is centrifuged for 20 min at 300 × g (Fig. 14.8). Seminal plasma, contaminating debris and leukocytes and the morphologically abnormal sperm, is carefully aspirated down to the pellet and discarded. The pellet along with some of the gradient is resuspended in 2 mL of sperm washing medium and centrifuged for 7 min at 300 × g (Fig. 14.9). Following centrifugation , the pellet is finally resuspended in 0.5 mL of sperm wash medium and examined for concentration and motility before cryopreservation.
Fig. 14.8
Double-density gradient wash procedure; separation of seminal plasma, abnormal non-motile sperm and viable motile sperm [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Fig. 14.9
HTF resuspended sample centrifuged to produce viable sperm pellet [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Sperm Preparation by the Swim-Up Method
This is another common technique for sperm preparation. Swim-up can be performed using either a pre-washed soft-spun pellet or liquefied semen sample. It is placed in an overlaying culture medium (sperm wash medium). Highly motile sperm swim to the top. In brief, the semen specimen is mixed with sperm wash medium (1:4 vol./vol.) and centrifuged for 10 min at 300 × g. After carefully aspirating the supernatant, the pellet is resuspended in 3 mL of the sperm wash medium. The sperm suspension is transferred into two sterile round-bottom tubes. After centrifuging the tubes for 5 min at 300 × g and kept at an angle of 45° for 1 h at 37 °C (Fig. 14.10). The angle and the use of round-bottom tube are important to increase the surface area and allow more motile sperm to swim to the surface. After incubation, the entire supernatant is carefully aspirated from the two tubes and pooled in a 15 mL conical centrifuge tube. Following another centrifugation, the clear supernatant is aspirated and the pellet resuspended in 0.5 mL of the sperm wash medium. After the initial sperm concentration and motility the sample can be cryopreserved.
Fig. 14.10
Swim-up procedure showing the separation of highly motile spermatozoa [reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved]
Effect of Cryopreservation on Sperm Characteristics
Cryopreservation damage or cryoinjury to the cells can be due to a combination of four factors: osmotic stress/dehydration, intracellular ice formation, cryoprotectant toxicity and oxidative stress [48, 49]. Glycerol in the cryoprotectant can result in cell toxicity. At very low temperatures, such toxic effects can be masked or diminished by slow metabolism of the cells. However at higher temperature such as at thawing, substantial sperm toxicity effects are evident when very high concentration is used. Some of the negative effects of sperm freezing and thawing are osmotic stress, extracellular and intracellular ice crystals resulting in cell injury, production of ROS which in turn has a negative effect on sperm motility, alteration in lipid phase, membrane integrity, mitochondrial function, DNA integrity, cell signalling and metabolism. In addition, sperm freezing can also result in apoptosis and necrotic cell death [50, 51]. Most of these negative effects occur during thawing stage. After thawing, these factors are responsible for a 25–75% loss of sperm motility, decrease in sperm cryosurvival and sperm DNA fragmentation [48, 49].
Motility is the single most affected parameter after cryopreservation [52, 53].The decline in motility is significant especially in patients who have poor sperm quality to begin with and is attributed largely to mitochondrial membrane damage resulting in a decrease in ATP production and poor sperm tail movement [43, 54, 55]. The differences in sperm membrane phospholipid, glycolipid and sterol content contribute to the poor motility [56]. Both osmotic and oxidative stresses have been shown to induce ROS in sperm after freezing and contribute to lethal or sublethal cellular damage [53, 57–59]. In addition, cryopreservation also results in DNA damage, especially DNA fragmentation that varies with the sample and is a result of oxidative stress-induced DNA damage [60–62]. Proteins play key roles in sperm metabolism, membrane permeability, flagella structure and motility, apoptosis, intracellular signalling, capacitation and fertilization that are altered after cryopreservation [63]. In addition, sperm proteome changes have been reported at every stage of the cryopreservation process. This ultimately impairs the fertilizing ability of the sperm, especially at the time of removing the sperm from storage and thawing at 23 °C [64].
Cancer Patients
Teratozoospermia is the most common (93.2%) abnormality among pre-treatment cancer patients [65]. An increase in chromosomal aneuploidy rate is seen in germ cells in men with testicular cancer and Hodgkin’s disease even before cancer treatment [66]. Both in healthy individuals and testicular cancer patients, viability and motility show a similar decrease after cryopreservation. A survival rate of only 44.8% with lowest odds of having total motile sperm count (TMC) above 5 × 106 was reported in men with testicular germ cell tumour (GCT) compared with controls and men with other cancers [67]. The lowest odds of successful intrauterine insemination was also seen in these patients. Non-seminoma germ cell tumour (NSGCT) is associated with higher post-thaw TMC than seminoma patients [68, 69]. Post-thaw TMC and cryosurvival have important clinical implications for couples who desire a pregnancy using ART . Sperm concentration of >5–10 × 106 sperm is predictive of successful IUI [70, 71]. Patients with TGCT are recommended to freeze a minimum of 15 vials (about 1 × 106/vial) before beginning their oncological treatment. This ensures availability of adequate sperm for two IUI attempts and sufficient sperm for use with IVF if both attempts fail [67].
Although semen quality is comparable in repeated ejaculates from cancer patients [72]; conflicting results have been reported between cancer stage and semen parameters [67, 73, 74]. Optimizing the post-thaw TMC and cryosurvival is therefore important [75]. Sperm of poor quality can be used for intracytoplasmic sperm injection (ICSI) where the availability of a single motile sperm is the only male factor determining successful fertilization. Success rates of IVF and ICSI treatment using cryopreserved sperm are almost as high as fresh semen. In cancer patients, using cryothawed sperm, the pregnancy rate per cycle and per couple in the IUI group was 11 and 32% while it was 37 and 68% in the ICSI group. ICSI is the preferred method of treatment for achieving pregnancies using cryopreserved sperm in cancer patients [76–81].
Freezing Testicular and Epididymal Aspirates
Patients with non-obstructive and obstructive azoospermia can also freeze their specimen obtained from the testis or the epididymis. For non-obstructive azoospermia, micro-testicular sperm extraction or micro-TESE is used to locate and excise the tubules that show spermatogenesis. Fine-needle aspiration or testicular sperm aspiration (TESA) is used to aspirate multiple testicular sites in case of obstructive azoospermia. Adequate number of sperm are required for IVF using TESA. Micro-TESE has the highest sperm retrieval rate compared to multiple fine-needle aspirate or TESE and the success rates are in the range of 50–60%. After mixing the aspirate with HTF, the mixture is centrifuged, and the pellets are resuspended with HTF. After mixing the suspension with an equal volume of cryoprotectant (e.g. Test Yolk Buffer), the sample aliquots can be loaded in the cryovials.
The procedure is somewhat different for sperm retrieval by testicular sperm extraction (TESE) from that used for testicular aspiration. In this case, tissue biopsies are obtained and shredded into small pieces using a sterile 25-gauge needle or fine scissor, mincer. Enzymatic digestion using type IV collagenase, trypsin, and trypsin inhibitor can also be used to allow the permeating cryoprotectant such as glycerol to fully penetrate testicular homogenate. Each tissue sample is carefully examined for the presence of motile spermatozoa using an inverted microscope. If no motile sperm are present in the testicular biopsy, further biopsies may be taken from the ipsilateral and contralateral testes until mature sperm are found. Conventional freezing of testicular sperm results in a sperm recovery rate of only 1% [82].
Microsurgical Epididymal Sperm Aspiration or (MESA) or Percutaneous Epididymal Sperm Aspiration or (PESA) is used to harvest epididymal sperm. PESA is effective in cases where the obstruction is at a site distal to epididymis. Compared to PESA, sperm retrieval is better with MESA; both show comparable fertilization and clinical pregnancy rates [83].
Sperm recovery using ICSI with larger cryoprotectant volume can be difficult with the conventional freezing techniques [84]. Different biological or non-biological carriers have been used to freeze microquantities of spermatozoa. The biological carriers can be empty zona [85–88] and Volvox globator algae [15, 89] and non-biological carriers include use of straws, mini straws [90, 91], high-security straws [92], cryoloop [91], ICSI pipette microdroplets [15], agarose gel microspheres [15, 93] and sleeper cells [94].
Fresh vs. Frozen Ejaculated Sperm and ART Outcomes
It is recommended that all men who are young adolescents should be offered semen cryopreservation [95, 96]. Several studies have found that the fertilizing capability of frozen sperm is lower than that of freshly ejaculated sperm [97, 98]. This is related with cryopreservation-related sperm dysfunction. However, identical pregnancy rates were reported between frozen and fresh ejaculated sperm using artificial insemination when sufficient number of progressively motile sperm were available in the post-thaw specimen and severe teratozoospermia and asthenozoospermia was not present [99].
Data is conflicting for conventional IVF ; while some investigators failed to show any difference in the fertility outcomes between cryopreserved ejaculated sperm and fresh sperm [100, 101], others have identified significant differences [102–104]. Advent and widespread use of ICSI has resulted in a tremendous modification in sperm banking practices as this procedure can overcome many limitations associated with sperm cryopreservation such as poor post-thaw sperm quality and sperm dysfunction. Kuczyński et al. conducted well-designed controlled trials and found no difference in fertilization rates between cryopreserved and freshly ejaculated sperm [105]. Meanwhile, the authors reported higher ongoing pregnancy rates per cycle with fresh sperm (23.7%) vs. frozen (35.2%) [105].
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