Chapter 14 The Process of Sperm Cryopreservation, Thawing and Washing Techniques Sajal Gupta, Rakesh Sharma, and Ashok Agarwal Introduction Reproduction is a key need which is central to human experience and drives quality of life. There is a growing list of conditions wherein there is a detrimental impact on fertility as a result of the disease itself or the therapeutic interventions directed towards the treatment of the disease conditions such as cancer or other extreme medical conditions. Sperm cryopreservation is used for these men, as they may desire to have children in the future, but current circumstances prevent the certainty that they will be able to conceive through traditional methods. This can be because of dangerous military deployment overseas, cancer or other toxic treatments which cause infertility, and many other situations. Sperm cryopreservation procedure involves the collection of the sample, freezing and long-term change storing to storage of the sample in extremely low temperature under liquid nitrogen at −196° C for possible use in the future to help achieve parenthood. All men who undergo treatments detrimental to repro- ductive function or face the challenge of their fertility being impacted by chemo- therapy or radiation therapy should exercise the option of semen collection for cryopreservation [1]. Cryopreservation of human semen is an important proce- dure for preservation of gametes in patients undergoing gonadotoxic treatment. Even some fertile couples can experience difficulties in conceiving due to geo- graphical or professional challenges and a general deterioration of male repro- ductive health over time is also an important indication for sperm banking. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have radi- cally advanced the treatment of male-factor infertility. As a result sperm cryo- preservation has become a successful treatment option [2]. ICSI requires a few S. Gupta, MD • R. Sharma, PhD • A. Agarwal, PhD (*) Department of Urology, Andrology Center, X-11, Cleveland Clinic, 10681 Carnegie Avenue, Cleveland, OH 44022, USA e-mail: [email protected] © Springer International Publishing AG 2018 183 A. Majzoub, A. Agarwal (eds.), The Complete Guide to Male Fertility Preservation, DOI 10.1007/978-3-319-42396-8_14 184 S. Gupta et al. viable sperm for successful fertilization of the oocytes selected based upon their competence [3]. Indications for Sperm Cryopreservation There are two groups of users for the sperm banking services: donor and directed client depositors [1]. Donors provide samples which can be used by other couples wherein the male partner has refractory male infertility and also could be utilized for same-sex lesbian partners. Directed client depositors freeze their samples exclu- sively for future use by their sexually intimate partner. Cancer Patients Every year more than 1.3 million patients are diagnosed with cancer in the United States. Over 44% of patients referred for sperm banking are cancer patients [1]. Patients receiving gonadotoxic treatment are at high risk for developing infertility [4]. Men with systemic (nonreproductive) cancers have relatively normal semen param- eters whereas men with testicular cancer [5–7] and lymphoma [8–10] have poor sperm parameters. Potential chromosomal aberrations in sperm exposed to chemo- therapy are compelling reasons for sperm banking before treatment [11]. Early sperm banking is recommended as 77.8% of patients are reported to be azoospermic after receiving cancer treatment [12]. Majority of the patients diagnosed with testicular cancer are in the reproductive age who can benefit from sperm banking [13, 14]. Travelling Husbands Geographical challenges where one of the partners is absent due to travel is an indi- cation for sperm banking as timed intercourse with ovulation is not possible. Cryopreservation of sperm allows the female partner to utilize cryopreserved sperm when she is ovulating [1]. Pre-vasectomy Men utilizing bilateral vasectomy as a contraceptive measures can elect to store sperm prior to the procedure. This helps keep their options open in the future if they go through some life-changing circumstances or demographic changes. 14 The Process of Sperm Cryopreservation, Thawing and Washing Techniques 185 Prior to ART Procedure About 12% of couples are infertile and 30–40% of these couples cannot conceive because the male partner has infertility issues or is azoospermic [15]. In such severe cases the couples may decide to use cryopreserved donor sperm. Extreme Medical Conditions Management of medical conditions such as autoimmune disorders, kidney disorders, ulcerative colitis or heart transplant and diabetes involves use of cytotoxic and immu- nosuppressive drugs. These drugs have the potential to suppress spermatogenesis [16]. Adolescents One in 1300 US males is a childhood cancer survivor and these adolescent cancer patients need to be offered fertility preservation services. The adolescent can collect a sample if puberty has set in. However, in adolescents who have not achieved puberty, there are limited experimental strategies such as spermatogonial stem cells autotransplantation [17]. The American Society for Clinical Oncologists also rec- ommends discussing fertility preservation options with adolescents. Emergency Fertility Preservation Emergency fertility preservation is defined as urgent need for sperm cryo- preservation in emergency situations such as scrotal trauma, either unilateral or bilateral, and testicular torsion. Scrotal exploration and emergency TESE is an option for these cases [18]. Posthumous Sperm Retrieval and Freezing Viable sperm can be obtained through posthumous sperm retrieval and freezing. There are reports of viable cadaver sperm obtained up to 36 h post-mortem. Fertilization rates ranging from 40 to 100% and singleton pregnancies have been reported with the use of posthumous sperm [19]. However use of posthumous sperm remains controversial due to ethical and legal concerns [20, 21]. 186 S. Gupta et al. Techniques of Sperm Cryopreservation Different cryoprotectants and cryopreservation methods have been used to preserve the viability of the sperm [22]. It is important to use a suitable cryoprotective agent to protect sperm cells during the freezing process and obtain good-quality sperm after thaw. All cryopreservation techniques have some detrimental effects on the overall sperm quality [23]. Slow Freezing Cryoprotectants function by permeating cells and lowering the concentration of electrolytes. Sperm cells are exposed to less osmotic strain with slow cryopreserva- tion. Although glycerol and egg yolk have been used, egg yolk provides superior post-thaw sperm quality compared with glycerol alone [24]. TES [N-Tris (hydroxy- methyl) methyl-2-aminoethane sulfonic acid, pK = 7.5] and Tris [(hydroxymethyl) amino methane] combined with fresh egg yolk, dextrose, and penicillin-streptomycin comprise the cryobuffer known as TEST-yolk buffer (TYB). It is the preferred cryo- protectant with higher longevity [23, 25]. TYB stabilizes sperm by altering phospholipid:cholesterol ratio and reducing damage by reactive oxygen species resulting in improved recovery of higher percentage of motile sperm, increased capacitation and sperm penetration [25]. The manual slow freezing method is used at our institution. It takes about 2–4 h to complete. It involves the gradual cooling of sample from room tempera- ture to –20 °C and the temperature is further lowered to –80 °C at the rate of 1–10 °C/min before plunging into liquid nitrogen at –196 °C. The patient/client depositor collects semen sample in a sterile collection cup by masturbation. The container is labelled with two identifiers. The sample is incubated at 37 °C for 20–30 min (Fig. 14.1). After liquefaction, the volume of the sample is measured and recorded. A note is made if any unusual viscosity, white blood cells or sperm agglutination is observed. 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. The percent motility is recorded as “pre-cryo motility %.” Using a sterile sero- logical 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 14 The Process of Sperm Cryopreservation, Thawing and Washing Techniques 187 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] 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] 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.