Quick viewing(Text Mode)

Recovery, Preparation, Storage and Utilization of Spermatozoa for Fertility Preservation in Cancer Patients and Sub-Fertile Men

Recovery, Preparation, Storage and Utilization of Spermatozoa for Fertility Preservation in Cancer Patients and Sub-Fertile Men

Utilization of spermatozoa for 150

Recovery, preparation, storage and utilization of spermatozoa for fertility preservation in cancer patients and sub-fertile men

Sajal Gupta, PhD1, Ashok Agarwal, PhD1, Reecha Sharma, PhD 1, Ali Ahmady, PhD 2,3

1Centre for Reproductive Medicine, Glickman Urological & Kidney Institute, & Obstetrics and Gynaecology & Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA 2Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH, USA 3MacDonald IVF and Fertility program, University Hospitals Case Medical Centre, Cleveland, OH

Abstract is an important part of an program for patients undergoing infertility treatments, fertility assurance for cases, and for fertility preservation due to cancer or other medical conditions. With recent developments in reproductive technology, even men with severely impaired sperm parameters can benefit from cryopreservation as procedures such as intra-cytoplasmic sperm injection (ICSI) require only a few sperm to achieve fertilization and pregnancy. 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. It is a highly effective method of protecting male fertility potential, and involves collection, freezing, and long-term storage of sperm. Based on the etiological condition of the patients, sperm can be collected by or by surgical retrieval from epididymis or testes. The option to bank sperm should be offered systematically to all patients who may benefit. However, this is not a standard of practice yet; it may be overlooked due to lack of physician awareness regarding the need for fertility preservation and the effectiveness of this option, and/or overestimating the limitations of poor baseline sperm quality leading physician to view cryopreservation as futile. Failure to offer cryopreservation ignores the only possible reproductive option available to certain patients.

J. Reprod Stem Cell Biotechnol 1(2):150-168 The authors have no potential conflicts of interest, whether of a financial or other nature Correspondence: A Ahmady2,3, PhD, HCLD; [email protected]; T: (216) 844-3317; F: (216) 201-4398

Sperm banking & cryopreservation outcomes. Recovery of spermatogenesis, Cryopreservation of human is an death, and anxiety regarding ART, financial important procedure used regularly for considerations, and uncertainty about long different purposes, including donor term prognosis are just a few reasons why and the preservation of patients who cryopreserve do not return for gametes in patients undergoing gonadotoxic infertility treatment (Hourvitz et al. 2008). treatment. Conception can be difficult even Men suffering with cancer, azoospermia, for some fertile couples for a variety of and other infertility problems have the option reasons and cryopreservation may be to cryopreserve their sperm. However, beneficial for them. Sperm banking allows cryopreservation has the ability to impair men to protect their future fertility. There is a sperm motility, vitality, and acrosome limited use of by integrity (Esteves et al. 2000a) . There is physicians in the majority of IVF programs. continuous research involving improvement This leads to an inability to improve care for of the various methods of cryopreservation patients that could benefit from this and cryoprotectants. technology (AbdelHafez et al. 2009). Physicians have certain doubts about the A. Relevance to modern medicine/ and justification of banking sperm before today’s world . Few patients (less than 5- Advanced assisted reproductive techniques 10%) who bank sperm before cancer help millions of people suffering from sexual treatment return for infertility treatments, and dysfunction, cancer and those undergoing about half of these patients have successful gonadal surgery. These techniques have

Utilization of spermatozoa for fertility preservation 151 given these people a chance to realize their any treatment which likely have adverse fertility potential. The sperm banks provide effect on the spermatogenesis process extended storage that allows sufficient time (Bonetti et al. 2009). Semen to perform screening on the donor. The cryopreservation should be performed developments made in intra-cytoplasmic before cancer treatment begins, and it is sperm injection (ICSI) and gamete isolation preferable that multiple samples are and maturation have promoted interest in preserved. All males of reproductive age cryopreservation of gonadal tissue which should consider banking semen samples appears highly promising for fertility before undergoing any type of treatments. Cryopreservation has numerous chemotherapy or radiation therapy, and advantages but also significant challenges physicians should always provide them with make it an active area of research (Bagchi the education they need to decide for or et al. 2008). The changing attitude towards against cryopreservation. sexuality has achieved a wider social phenomenon that has changed the behavior Sperm banking can be a difficult subject to as well as ideas concerning reproduction discuss with young patients and their and associated techniques (Mori 2008). because of sensitive topics such as developing sexuality, the grief associated B. Need for increased awareness by with facing infertility as a side effect, and Oncologists: as a means of collecting a Oncologists need to know about the regional sample, but it is still very important to availability of gamete cryopreservation preserve the reproductive future of the facilities. Physicians also need to be willing patient if possible (Menon et al. 2009a). This to discuss the issue of cryopreservation with study suggested that the majority of the patient and his family. In the era of ICSI physicians and about half of the patients when only a few sperm are needed to preferred to have initial discussions about achieve fertilization and pregnancy, even sperm banking without the patient’s parents men with severely impaired sperm present (de Vries et al. 2009). Semen parameters will benefit from sperm cryopreservation is the standard of care for cryopreservation and should be encouraged these individuals. Failure to offer this option to do so (Hourvitz et al. 2008). There is ignores the patient’s only reproductive limited use of cryopreservation by urologists option (Hourvitz et al. 2008). and gynaecologists in the majority of IVF programs. There is a lack of information Indications of sperm banking regarding the effectiveness of gamete A. Couples who can benefit from sperm cryopreservation and a lack of agreement on banking the best universal method. Unfortunately, Couples undergoing fertility treatment can this is a missed opportunity to improve care benefit from cryopreservation if the male for patients that could benefit from this partner is not available at the time of the technology. ovulation process. The success rates with cryopreserved sperm for intra-uterine C. Need for increased awareness among insemination (IUI) and ICSI cycles are patients similar to those with fresh sperm. The There is a lack of education/counselling by recommendation is to have the male the health care professionals (Hallak et al. partners with oligoasthenzoospermia to 1999a). It is of crucial importance that all bank multiple samples so that the samples newly diagnosed male cancer patients be can be used for repeat cycles in case of advised to cryopreserve their sperm at the failed cycles of IUI or ICSI. earliest stage and most importantly before starting treatment. Although many cancer i. Absent male partner patients have poor pre-treatment semen Fertile couples may use sperm quality, most have suitable sperm for cryopreservation for a number of reasons. If freezing with good expectations for sperm either the male or female partner is often survival. All young males 12 years of age or absent, for example, when travelling for older should be offered the opportunity to business, the couple may find conception bank their sperm prior to administration of difficult. It may be hard for the couple to

Utilization of spermatozoa for fertility preservation 152 coincide intercourse with ovulation. reducing the morbidity associated with Cryopreservation of sperm may be gonadotoxic treatments (Hallak et al. convenient for the couple, allowing the 1999b). Hodgkin’s disease, testicular woman to receive the male’s sperm in a cancer, leukaemia, and non-Hodgkin’s clinical setting when she is ovulating. lymphoma are the most common malignancies seen in the male reproductive- ii. Participating in ART age group (Hourvitz et al. 2008). Analysis of About 12% of couples are unable to covariance of semen parameters showed conceive after one year of unprotected the greatest impairment in prostate cancer intercourse and are therefore considered patients. Testicular cancer patients had infertile (Eisenberg et al. 2009). About 30- relatively lower sperm counts but relatively 40% of these couples cannot conceive good motility. Men with lymphomas and because the male partner has infertility other systemic (non-reproductive) cancers issues, and 10% of male factor infertility is have relatively normal semen parameters. caused by azoospermia. In the most severe Testicular cancer (Audrins et al. 1999; cases of , couples may decide Berthelsen 1984; Botchan et al. 1997b) and to use a sperm donor (AbdelHafez et al. lymphoma (Botchan et al. 1997a, Chapman 2009). et al. 1981) have been associated with impaired sperm quality. B. Cancer patients The various gonadotoxic treatments can Padron et al. (1997) have shown similar have differential effects on spermatogenesis in men with Hodgkin’s and sperm quality in patients with diverse disease, leukaemia, and testicular cancer. types of cancers. A European study Several of the most common malignancies observed that the incidence of testicular in men of reproductive age have good long- cancer has doubled in the past 20 years, term survival rates, for example, testicular and the incidence of germline testicular cancer and Hodgkin’s disease (Howell and cancers increased drastically from 1967 to Shalet 2001). However, antineoplastic 1987, as well as 1987 to 1996 (Lacerda et therapy is associated with significant al. 2009). Between 15-30% of male patients morbidity, and testicular dysfunction is undergoing gonadotoxic treatments do not among the most common long-term side regain their fertility (Menon et al. 2009a). effects of cytotoxic chemotherapy in men. Approximately 90% of men with testicular cancer ranked fertility as an important issue The degree to which testicular function is after cancer treatment (Bonetti et al. 2009). affected is dose and agent dependent Both male and female cancer survivors (Palermo et al. 1992). Alkylating agents report a large degree of stress regarding (e.g., cyclophosphamide and busulfan) and their own ability to reproduce (de Vries et al. ionizing radiation frequently induce 2009), which can have a high impact on azoospermia, rendering the patient infertile. one’s quality of life. Each year, Another major reason to freeze sperm approximately 1.3 million patients are before treatment is the concern for potential diagnosed with cancer in the United States, chromosomal aberrations in sperm exposed with an average 5-year survival rate of 60%, to chemotherapy (Lass et al. 2001). resulting in about 9.8 million cancer Although no increase in malformation rate survivors. Today, the cure rate for testicular has been reported in children born to cancer, Hodgkin’s disease, lymphoma, and patients who have had chemotherapy or leukaemia can be as high as 90% (Bonetti et radiotherapy, the available data and follow- al. 2009). up are still limited and these children should be closely monitored. Semen parameters in Patients receiving radiotherapy are at high patients with lymphomas and all other risk for developing infertility, and cancer systemic malignancies were associated with surgery can reduce sperm concentration better semen profile than in patients with and cause erectile dysfunction or dry testicular or prostate carcinoma (Hourvitz et ejaculation. Full recovery can be achieved in al. 2008). After receiving cancer treatment, the majority of these patients; so many 77.8% of patients became azoospermic, recent efforts have been concentrated on

Utilization of spermatozoa for fertility preservation 153 which emphasizes the importance of early successive multiple toxicity, like bone cryopreservation. marrow transplantation (de Vries et al. 2009). Infertility is a major sequel of cancer and/or its therapy. The quality of spermatozoa in Currently there is a high incidence of men diagnosed with cancer is suboptimal, malignant testicular tumors, lymphatic, and even prior to the initiation of hematopoietic tissue origin tumors among chemo/radiotherapy. Lower sperm quality in men of reproductive age (Crha et al. 2009). patients with testicular malignancy can be The high remission rates have caused an explained by the fact that genital tumours increase in recent efforts to reduce the exert local negative effects. Sperm morbidity associated with gonadotoxic concentration was significantly lower in treatments (Hallak et al. 1999b). patients with testicular malignancy Radiotherapy patients are at high risk for compared to those with systemic developing infertility, and cancer surgery can malignancy and healthy proven fertile reduce sperm concentration, cause erectile donors (p<0.001, p<0.05, respectively). dysfunction or dry ejaculation (Bonetti et al. Motility was found to be significantly lower in 2009). patients with testicular and systemic malignancy compared to healthy proven C. Before prostate/testicular fertile donors (p<0.001: Williams et al. surgery/biopsy 2009). In a study with 2680 subjects the The testicular tissue can be cryopreserved average age of men with testicular cancer at the time of surgery for future use in was 29.9 years prior to therapy that signifies treatment of infertility. Mature spermatozoa the importance of cryopreservation in this extracted from frozen thawed testicular target population. Men with testicular biopsies have been used in ICSI (Bagchi et cancer usually have a lower sperm count al. 2008). A study was conducted in patients and motility due to the tumour growth. with prostate, bladder or kidney cancer to Infertility is clearly associated with therapy, find out whether these cancers were an and chemotherapy doses (Schrader et al. independent predictor of the patients wish 2001). The majority of patients develop for sperm banking before any surgical or azoospermia 12 weeks after beginning of nonsurgical therapy which may have a chemotherapy. potential adverse effect on male fertility. The study findings showed that having the Chemotherapy targets cells outside the G0 therapy non-germ cell urological cancer phase, destroying proliferating (NGCUC) was independently associated spermatogonias (Bonetti et al. 2009). The with the wish for cryopreservation (Salonia majority of chemotherapy patients develop et al. 2009). azoospermia during treatment and it is difficult to predict if and when D. Severe oligozoospermia or low quality spermatogenesis will recover. This tends to sperm be dose dependent; while patients receiving Azoospermia may occur in some healthy low doses of these agents may recover men, and natural fertilization may be spermatogenesis within 12 weeks of impossible for them. However, completing chemotherapy, more than 50% cryopreservation and IVF may allow these of patients receive high doses which might men to children. The recovery of contribute to the 15-30% of all patients who viable is comparatively low with less remain sterile in the long term. It is than 60% of cells retaining motility on estimated that up to 15% of male patients thawing. This loss of viability becomes a will already be azoospermic before major issue in case of oligozoospermic undergoing any type of treatment. Semen samples. For these cases there is a growing cryopreservation should be performed need to store low numbers of sperm by before cancer treatment begins, and it is developing improved freezing techniques. preferable that multiple samples are The use of frozen-thawed testicular biopsies preserved (Bonetti et al. 2009). Patients who in ICSI is very helpful in patients with are most at risk are those who undergo a obstructive azoospermia with normal treatment modality encompassing spermatogenesis. (Bagchi et al. 2008).

Utilization of spermatozoa for fertility preservation 154

ejaculatory problems or if they are an adolescent. The various issues related to E. Ejaculatory dysfunction are discussed below. Erectile dysfunction and anxiety issues may prevent a couple from successfully i. Privacy conceiving, so cryopreservation and IUI can Sperm donation raises a number of ethical allow these couples to conceive in a clinical questions for the sperm donors. Since setting. decades there have been questions raised on anonymous sperm donation. Donors F. Prior to vasectomy usually have no legal or financial Indications for sperm banking other than for responsibility for the child. But there are infertile men are collectively included under exceptions to the case as seen from time to the term “special storage” which includes time in many legal situations. The ethical storage as insurance before vasectomy dilemma arises about the responsibility of (pre-vasectomy storage) and storage before the child and also in certain cases where treatment that could damage sperm there is a need to find out about genetic production, such as cytotoxic therapy or conditions. radiation therapy (medical storage) (Audrins et al. 1999). A patient undergoing ii. Difficulty providing semen sample vasectomy is offered sperm banking prior to Reasons could be anejaculation, vasectomy. The issue to consider is sperm psychogenic ejaculation, spinal cord injury, freezing and storage (cryopreservation) as a retrograde-ejaculation etc. The prevalence reasonable alternative to vasectomy of premature ejaculation varies from 8 to reversal in the unlikely event that the 30% for all age groups (Corona et al. 2010). individual wishes to father children in the future. The reversal of vasectomy procedure I. Cryptorchidism and genetic disorders is expensive and requires hospitalization With the help of cryopreservation germ cells and utilizes time. It is therefore far more are harvested at an earlier stage of cost-effective to cryopreserve semen before development for further maturation in vitro. the vasectomy, which then can be stored Over the past decade, developments in indefinitely at little cost (Audrins et al. 1999). microsurgical techniques and advances in The availability of ICSI technology has made ART allowed more than 50% of patients with cryopreservation of sperm during vasectomy Klinefelter’s syndrome (KS) to have their reversals possible. own children through the combination of microsurgical testicular sperm extraction G. Men with high-risk occupations (TESE) and use of freshly retrieved sperm The exposure to environmental factors air for IVF. However, this technique requires an pollution, pesticides, phthalates and PCB expensive surgical procedure and hormonal has been reported to affect semen quality stimulation of a female partner despite the (Jurewicz et al. 2009). The occupations uncertainty of sperm recovery from testis. related to the use of chemicals, pesticides, etc are considered high- risk and men Surgical sperm retrieval engaged in these high risk work areas Surgical sperm retrieval techniques are should be offered sperm banking as an applied for azoospermia cases which have option. either obstructive or non-obstructive. Percutaneous epididymal sperm aspiration H. Issues related to sperm donation (PESA) and microsurgical epididymal sperm Cryopreservation is an important tool that aspiration (MESA) are used for obstructive preserves fertility for men with cancer and is case, while testicular sperm aspiration akin to providing these individuals “fertility (TESA) and testicular sperm extraction insurance”. Patients are concerned about (TESE) are applied for non-obstructive the quality of life issues related to cancer cases. treatment and want to preserve the sperm for future fertility. However the cancer A. Epididymal sperm retrieval techniques patients may have difficulty in providing Epididymal sperm can be retrieved by two semen samples due to privacy issues or methods of percutaneous epididymal sperm

Utilization of spermatozoa for fertility preservation 155 aspiration and microsurgical epididymal (testicular sperm extraction TESE). sperm aspiration. Recently, however optical loupe magnification TESE (microdissection TESE) i. Percutaneous epididymal sperm was applied in retrieving sperm from non- aspiration (PESA) obstructive azoospermia cases (Mulhall et PESA is performed without surgical scrotal al. 2005). All procedures are performed exploration. It does not require an operating under anesthesia (general, or local). microscope or expertise in microsurgery. To Generally, the scrotum is opened via a perform, a butterfly needle (attached to a 10- median raphe incision and all layers are cut 20ml syringe) is inserted into the caput until there is full exposure of the testis. epididymis and tip of the needle is gradually moved within the epididymis until clear or i. Testicular Needle aspiration opalescent fluid seen in the needle tubing. Testicular needle aspiration was initially The procedure is repeated until adequate used for diagnostic purposes and later to amount of epididiymal fluid is retrieved (Craft establish the likelihood of sperm retrieval for et al. 1995). The aspirate is then flushed into testicular ICSI cases on the day of oocyte a sterile tube before sending to lab for retrieval (Fasouliotis et al. 2002; Turek et al. evaluation and processing. 1999). Different techniques have been described with variations in the needle ii. Microsurgical epididymal sperm diameter and the number of testicular aspiration (MESA) punctures (Donoso et al. 2007). Lewin et al. It is performed under anesthesia. After (1996) successfully performed testicular fine opening the tunica vaginalis and exposing needle aspiration (TFNA) to retrieve sperm the epididymis, single epididymal tubules from a patient with maturation arrest and are identified under operating microscope. elevated gonadotrophins. The tubules are then punctured and the Testicular needle aspiration is usually effluent aspirated into an aspiration device performed in 3 different locations: in the (syringe). Sequential aspirations are centre of the testis and in the upper and performed until optimal quality sperm are lower poles; with the aim of aspirating obtained (Schlegel et al. 1994; Tournaye et testicular tissue from the depth of the testis. al. 1994). Best quality sperm are found in Upon exposure of testes, the needle is the proximal epididymis close to the testis. inserted into the centre of the testis and The aspirates are then sent to lab for negative pressure is applied. While evaluation and processing. maintaining negative pressure, the needle is partially withdrawn and inserted again at B. Testicular sperm retrieval techniques different angles. The sampling is performed It has been shown that mature spermatozoa using a needle biopsy gun that enabled a can be found in only part of the testes of controlled and accurate sampling as well as non-obstructive azoospermic man. In the creation and maintenance of a examining the testes of infertile men, Levin substantial negative pressure during the (1979) found a mixed histological pattern of procedure. A separate 20-mL syringe germinal cell aplasia and minute foci (focal) containing 0.5 ml of culture medium and an spermatogenesis. A similar histology of side 18 gauge needle are used for each sample. by side presence of different patterns of The aspirated samples are transferred focal spermatogenesis and Sertoli-cell immediately to the laboratory for sperm syndrome was observed in non-obstructive search and isolation (Hauser et al. 2006). azospermic men (Devroey et al. 1995; Gil- Salom et al. 1995). Therefore, the ii. Testicular sperm extraction (TESE) performance of multiple focal testicular For TESE, the tunica albuginea is incised sperm retrieval was recommended to ensure transversely at 3 locations of centre, upper, the presence of sperm in testicular sample and lower poles in each testis. The testis is (Hauser et al. 1998). then gently squeezed and the protruding The most common methods for retrieving tissues are excised, each weighing the testicular sperm are testicular sperm approximately 50 mg. The biopsy material is aspiration (TESA: needle/fine needle placed in culture medium and transferred aspiration), and open testicular biopsy

Utilization of spermatozoa for fertility preservation 156 immediately to the laboratory for sperm cell importance of having children, emphasis on isolation (Hauser et al. 2006). the benefits of banking and addressing possible obstacles such as cost, Microdissection TESE is an advanced misconceptions or cultural and other factors version of TESE that applies microsurgical (Achille et al. 2006). techniques for sperm retrieval. In this It is very surprising to see that in spite of a technique, individual seminiferous tubules high risk of developing infertility and the can be seen under the surgical microscope available option of sperm banking, still very allowing the identification of active few patients go ahead with banking of their spermatogenesis sites (Schlegel 1999). This sperm (Achille et al. 2006). strategy could facilitate the removal of smaller amounts of testicular tissue, which A. Priority becomes crucial in testicular atrophy cases Sperm banking is not usually a priority for and minimize the chance of vascular injury patients that have already completed their as vascular regions of the tunica albuginea family, for those that who do not want to during the opening could be identified have children, and for those patients who (Donoso et al. 2007). think that they maybe delaying their cancer treatment by selecting the option of sperm C. Laboratory preparation of TESE and banking. Some of the patients are too young TESA sample to understand the impact of gonadotoxic Upon receipt of the biopsy tissues, they are therapy or surgery (Achille et al. 2006). shredded into small pieces with sterile 25 gauge needle or fine scissors. The presence B. Cost of spermatozoa is assessed using an A Presumed high cost for the procedure is a inverted microscope. The effluents as well major factor inhibiting patients from electing as the shredded biopsy tissue can be to bank sperm (Bonetti et al. 2009). The cost centrifuged and the pellet re-suspended in of banking of sperm is not covered by the culture medium, incubated in few droplets insurance companies and may be a very under mineral oil for ~1 h prior to selection of important factor for rejection of banking by spermatozoa. Motile spermatozoa usually low income patients. migrate to the edge of the drop (Craft and Tsirigotis 1995; Nijs et al. 1996). C. Time interval Alternatively, original shredded specimen The urgency to start chemotherapy as soon can be incubated in droplets under the oil as cancer diagnosed is one of the major without centrifugation prior to selection of factors that prevents a number of patients to sperm. The selected spermatozoa can be go for sperm banking. The risk for infertility washed in PVP droplet before proceeding to following treatment for cancer is higher in injection into oocytes. adolescent males. Therefore treatment exposures for them should be carefully FACTORS PREVENTING INDIVIDUALS assessed before initiating therapy. Fertility FROM SPERM BANKING preservation by sperm banking should be A qualitative study on 20 cancer survivors part of the initial treatment discussion with and 18 health care professionals conducted family and the patient (Hobbie et al. 2009). in-depth interviews to examine their perspectives on factors that facilitate or D. Lack of information hinder sperm banking. The data collected A study was done to survey oncologists in was analyzed using a mixed approach and a different practice settings to determine their three-step process of data reduction, data knowledge, attitudes, and practices display and conclusion drawing and regarding referring patients to bank sperm verification. The study reports several before cancer treatment. The study found factors that have an impact on Sperm that ninety-one percent of respondents Banking. The findings in the study agreed that sperm banking should be recommended that effective promotion of offered to all men at risk of infertility as a sperm banking involves adequate result of cancer treatment. Forty eight communication regarding the severity and percent of physicians did not mention the risk of infertility, assessment of the option to less than a quarter of eligible men.

Utilization of spermatozoa for fertility preservation 157

The reasons for lack of discussion and non dissemination of information by physicians Most countries require a mandatory formal on sperm banking were lack of time for the written consent for sperm donation. Many discussion, perceived high cost, and lack of ethical issues are raised regarding the rights convenient facilities. HIV-positive men, and obligations of the mother, the husband, patients with a poor prognosis, or having and the child. Ethical questions arise about aggressive tumors would be less likely to be duties and rights of the toward a child offered sperm banking (Schover et al. 2002). who is not their biological offspring (Meirow The staff at the oncologist’s office can and Schenker 1997). educate the patient thus reducing the amount of physician time spent in explaining F. Psychosocial issues with sperm sperm banking. Self help books can be banking – anxiety and emotional stress provided to patients that discuss cancer, It is a life crisis situation for many young male infertility, and sperm banking. The individuals affected by cancer. The cancer sperm banks are very easily found on diagnosis and the threat of infertility both various websites like www.sperm- cause a tremendous stress on these banks.com. The availability of home kits for individuals (Tschudin and Bitzer 2009). sperm collection and express shipping to the Schover et al. (1999) have elucidated makes it more convenient for several causative factors for psychological the patients to collect the sample in the stress in cancer patients such as distress privacy of their homes (Schover et al. 2002). related to future infertility, concern about Patients who have been properly advised quality of life issues, patients with distress about the high risk of infertility associated due to inheritable cancer and lack of with chemotherapy regimens for testicular knowledge regarding various risk factors cancer or Hodgkin’s disease are more likely associated with the gonadotoxic treatment to utilize sperm banking (Achille et al. 2006; (Schover 1999; Tschudin and Bitzer 2009). Schover et al. 2002). These factors need to be acknowledged by health care professionals and utilized in Another study observed that only 47% of proper care and treatment of these patients. oncologists always or often refer their patients to an REI or infertility specialist. The Screening of client depositors prior knowledge about banking is high among to sperm banking oncologists, but still there are a few Both male and female cancer survivors questions regarding fertility and cancer that report a large degree of stress regarding are not well answered such as the ability to their ability to reproduce, and its high impact determine an individual’s risk. Future efforts on quality of life (de Vries et al. 2009). are required to include additional training for Approximately 90% of men with testicular health care providers regarding this. The cancer ranked fertility as an important issue limited amount of time to discuss the cancer after cancer treatment (Bonetti et al. 2009). diagnosis and treatment plan as well as to 15-30% of male patients undergoing deal with the psychosocial issues of a newly gonadotoxic treatments do not regain fertility diagnosed patient is a major barrier. later (Menon et al. 2009b), so it is crucial to Therefore it is essential that nurses or social discuss sperm banking with all males of workers be trained to initiate discussions reproductive age. Physicians need to be about sperm banking. (Quinn et al. 2009). aware of the availability of gamete cryopreservation facilities and be willing to E. Religious or ethical concerns about discuss this issue with patients. Surveys sperm banking show that the lack of timely information is The practice of sperm donation is opposed the most common reason for not banking by many religions and hence the option of sperm (de Vries et al. 2009). sperm banking is not accepted by some infertile couples due to religious reasons. Screening is done in order to reduce the risk There is strong influence exerted on the civil of passing on sexually transmissible authorities in the field of reproduction and infections through insemination. The patient artificial reproduction by many religious is tested for sexually transmissible authorities. infections. Some banks also get an HIV risk

Utilization of spermatozoa for fertility preservation 158 assessment form; a personal and family of the sedimentation gradient and penetrate health history form; a medical examination the boundary quicker than poorly motile or (which must include a genital examination immotile sperm, enriching the soft pellet at for indications of sexually transmissible the bottom with highly motile sperm. infections); and blood testing to identify The most widely used gradient substance blood type and Rh factor. These for assisted reproduction has been the examinations must be completed no more polyvinylpyrrolidone (PVP) - coated silica than twelve months prior to the first storage particles called Pecoll. It was withdrawn appointment. from the market because of the risk of contaminations with endotoxins (Andersen The blood tests include HIV-1/2, HTLV-1/2, and Grinsted 1997; De Vos et al. 1997; hepatitis B, hepatitis C, syphilis, and in some Scott and Smith 1997), possible membrane cases CMV. Genetic Testing of the client alteration (Arcidiacono et al. 1983; Strehler sperm depositor or sperm donor is required et al. 1998) and inflammatory responses by certain states such as New York. induced by the insemination of sperm contaminated with Percoll. TECHNIQUES OF CRYOPRESERVATION A. Preparation and selection The replacement products for Percoll® are mechanisms prior to banking Nycodenz (Nyegaard & Co., Oslo, Norway), Swim up and density gradient are the two IxaPrep® (MediCult, Copenhagen, widely used methods for sperm wash and ), SilSelect® (FertiPro N.V., processing. Sperm washing techniques Beernem, Belgium), PureSperm® (NidaCon separate ejaculated spermatozoa from the Laboratories AB, Gothenburg, ) or seminal environment, and eliminate dead Isolate® (Irvine Scientific, Santa Ana, CA, spermatozoa along with exfoliated epithelial USA). These replacements were found to cells, cellular debris, leukocytes, and produce populations of highly motile amorphous material (Berger et al. 1985). spermatozoa with better yields and survival The swim-up method involves centrifugation than either swim-up or Percoll® gradients of a semen sample into pellets followed by from oligozoospermic and covering with culture medium; allowing the asthenozoospermic semen samples spermatozoa with better motion (Gellert-Mortimer et al. 1988; Mortimer characteristics and motility to swim up into 1994). Compared to the swim-up procedure, the culture medium to be selected for Percoll® replacement seems to be superior cryopreservation (Esteves et al. 2000b). In in zona-free hamster egg penetration test comparison to untreated specimens, swim (Serafini et al. 1990). Thus, this technique up cryopreserved sperm have been shown has clearly great potential in the preparation to exhibit faster velocity and progression, of motile spermatozoa from poor quality higher percentages of intact acrosomes, semen. increased ability to undergo acrosome reaction and, better performance in the Magnetic-activated cell sorting (MACS) is sperm penetration assay after thawing another preparation technique that has been (Esteves et al. 2000b; Russell and Rogers shown to select motile, viable, 1987). morphologically normal spermatozoa displaying higher cryosurvival rates and Density gradient centrifugation comprised of subsequent fertilization potential (Grunewald either continuous (Bolton and Braude 1984) et al. 2001; Grunewald et al. 2006; Said et or discontinuous (Pousette et al. 1986). In al. 2005). MACS uses annexin microbeads, continuous gradients, there is a gradual a phospholipids-binding protein with a high increase in density from the top of the affinity for phosphatidylserine (PS)- an early gradient to its bottom, whereas there is a marker of apoptosis, to label and remove clear boundary between each layer in apoptotic spermatozoa (Said et al. 2008). discontinuous gradient method. The Although this method may offer more ejaculate is placed on top of the density advantages over the other techniques by gradient, and centrifuged for 15–30 minutes. selecting genetically intact, non-apoptotic During this procedure, highly motile sperm it is still experimental. spermatozoa move actively in the direction

Utilization of spermatozoa for fertility preservation 159

B. Slow Freezing (vitrification) was developed by direct Slow freezing is based on the principal of plunging of a sperm suspension I nto liquid dehydration, where equilibration is achieved nitrogen (Isachenko et al. 2003; Nawroth et by combination of low cryoprotectant al. 2002). After storage, warming is achieved concentrations and slow rate of cooling, by direct melting of the frozen suspension. allowing dehydration to occur during cooling. This is a simple, straightforward approach It is the method of choice for preserves the motility and fertilizing ability of cryopreservation of human spermatozoa. In the spermatozoa. The improved results over this method, semen samples (raw and the conventional slow freezing (ice- washed) are diluted by drop-wise addition of equilibrium) may be attributed to omitting the freezing medium with continuous permeable cryoprotectants, thus preventing uniform mixing to a final ratio of 1:1 (volume- the lethal effects of osmotic shock to-volume). They are then loaded into straws (Isachenko et al. 2004). Vitirifcation of sperm or transferred into cryovials before exposure is relatively new technique and it is not to –20 °C for 15-30 minutes, followed by standardized yet to be implemented exposure to –79 °C for another 15-30 clinically. minutes before placing in liquid nitrogen for storage (WHO 5th Ed). This slow rate can be Advantages and disadvantages of achieved by using a programmable freezer various cryoprotectants or manually in liquid nitrogen vapour. Since Polge & Rowson (Polge 1952) first successful report of glycerol as Various techniques have been proposed for cryoprotectant for bull spermatozoa freezing of the sperm for surgically retrieved cryopreservation, there have been many cases where a few sperm are retrieved. One reports on the cryopreservation of approach is to inject isolated sperm cells spermatozoa of various species including into a hamster zona pellucida which is then horse (Nishikawa 1975) , pig (Pursel and placed in a straw between two air bubbles to Johnson 1975), sheep (Colas 1975), dog facilitate location of the cells after thawing. (Seager and Fletcher 1973), rabbit (Fox (Cohen et al. 1997; Hsieh et al. 2000). 1961), and man (Bunge and Sherman Others suggested the freezing of the minute 1953). It is the most widely used and sample under a layer of paraffin oil with successful cryoprotectant for human sperm. glycerol (Craft and Tsirigotis 1995). Romero A final concentration of 7.5% has been et al. (1996) described a frozen “testicular shown to be an optimal concentration of pill” composed of a mixture of some sperm glycerol for freezing solution. Egg yolk on and testicular tissue. the other hand, which is not a cryoprectant itself and often used in combination with C. Vitrification glycerol, seems to confer improved sperm Vitrification is preservation at extremely low plasma membrane fluidity, resulting in temperatures without freezing. Freezing improvement in cryo-survival (Hallak et al. involves ice crystal formation, which 2000). damages delicate organelles. Vitrification instead involves the formation of a glassy or Prins and Weidel 986 compared eight amorphous solid state which, unlike different cryopreservatives, concluding freezing, is not intrinsically damaging even sperm frozen with egg yolk buffer to the most complicated living systems. In demonstrated the highest post-thaw this process the ice formation during cooling survival. Mahadevan and Trounson (1983) is inhibited by high concentration of viscous developed a modified Tyrode’s medium solutions which produce glass-like state at containing 7.5% glycerol referred to as low temperatures. However, because Human Sperm Preservation Medium spermatozoa are of small size and contain (HSPM), demonstrating high little cytoplasm or water they are sensitive to in comparison with egg yolk-citrate-glycerol cryoprotectant (Gilmore et al. 1997; Mazur medium with no difference in post-thaw et al. 1981). motility and viability.

Recently a new technique of ice- and In comparing three cryopresevatives of cryoprotectant-free cryopreservation TEST yolk, glycerol, and HSPM, Centola et

Utilization of spermatozoa for fertility preservation 160 e. (1992)l demonstrated that HSPM had the to structural integrity of sperm that occurs best recovery in regards to concentration during cryopreservation, but are most likely and motility, while glycerol had better not responsible for DNA damage in sperm. recovery rate of progressive velocity than TEST yolk. These data suggest that HSPM B. Acrosomal integrity is a superior cryopreservative based on Zona-free hamster oocyte (ZFHO) post-thaw recovery of motile sperm, penetration assay evaluates the ability of a confirming earlier report of Mahadevan and sperm population to capacitate, acrosome Trounson (1983). react, bind and penetrate the membrane of an oocyte lacking a zona pellucida. When The effect of cryopreservation on acrosomal stimulants such as TEST-yolk sperm characteristics buffer are used to treat the sperm, the ZFHO A. DNA stability penetration assay correlates well with IVF Semen cryopreservation has been reported success. Poor results are expressed when to induce DNA damage. The exact low percentage of oocytes is penetrated. mechanism of the effect on DNA integrity is Poor results indicate that a male patient is not known. It has been proposed to be unlikely to impregnate his partner without induced by causing a rise in oxidative stress the use of ICSI. in the semen. Stimulation of caspases and The acrosome is an organelle which apoptotic mechanisms has been proposed facilitates the passage of the spermatozoa by some literature reports. This mechanism through the zona pellucida of the oocyte just has been refuted by other reports where prior to fertilization. Studies have shown that addition of caspase inhibitors to the acrosome is affected by cryopreservation cryopreservation medium did not prevent more severely than any other apoptosis. The addition of the caspase organelle(Ozkavukcu et al. 2008). Acrosome inhibitors had no significant effect on the is characterized by a fragile membrane post thaw motility. which is susceptible to changes in Cryopreservation can cause and exacerbate osmolarity and physical or chemical DNA fragmentation in spermatozoa conditions, which are extreme in case of (Thomson et al. 2009). Some studies cryopreservation of gametes. indicate that cryopreservation can increase Cryopreservation of sperm can lead to inappropriate chromatin condensation in acrosomal abnormalities, such as cracks or peelings, due to low temperatures which can human sperm (Yildiz et al. 2007). DNA 2+ integrity can be determined by several increase cytoplasmic Ca levels, methods. These methods include TUNEL, a capacitation-like reactions, ionic leakage, direct measure of DNA damage, and Comet, and exocytosis of acrosomal content an electrophoresis assay that evaluates (Ozkavukcu et al. 2008). how well DNA is packed within the nucleus (Bakos et al. 2008; Yildiz et al. 2007). C. Motility and viability The cryopreservation survival rate (%) is Sperm chromatic structure assay (SCSA) is calculated by dividing post-thaw motility by another method which measures the extent pre-freeze motility and multiplied by 100. A of DNA denaturation (Kobayashi et al. study reports on ART outcomes which 2001). This method allows one to decide analyzed 118 male cancer survivors whether the DNA within a sperm cell is undergoing 169 IVF-ICSI cycles(Hourvitz et structurally intact and normal or abnormal. al. 2008). Analysis of covariance, showed Oxidative damage caused by reactive that type of cancer was significantly oxygen species is suspected to play a role in associated with post-thaw semen cryo-injury to sperm DNA. Antioxidants such concentration (P<0.0001) and with total as genistein (a plant-derived phyto- motile sperm count (P<0.0005). All semen estrogen) and those found in native semen parameters were found significantly better in protect sperm from oxidative stress and lipid case of lymphomas as compared to all other peroxidation, and thereby reduce DNA systemic malignancies. fragmentation (Thomson et al. 2009). Increase in the activation of the intrinsic Furthermore, the effect of cancer type on apoptotic cascade might result from insults post-thaw semen parameters was not

Utilization of spermatozoa for fertility preservation 161 influenced by the cryopreservation storage have antioxidant properties that allow for the interval and the age of the patient at stabilization of the acrosomal membrane diagnosis. Post-thaw motility is reduced in and maintain the spontaneous acrosomal cryopreserved semen samples, and reaction. Pentoxifylline has beneficial depends on the pre-freeze motility of the effects of spermatozoa prior to semen sample (Ozkavukcu et al. 2008). cryopreservation and is proposed to improve Most studies indicate that viability and the fertilization ability of cryopreserved motility, the more important sperm spermatozoa. parameters determining independent fertilization capacity, are reduced by 50% Reasons for non utilisation of banked between the pre-freeze and post-thaw samples semen samples. It is likely that much (but The 95% of banked sperm samples are not not all) of the reduced motility is a direct utilized. The common reasons for the non- result of reduced viability caused by damage utilization could be death of the patient, to cell membrane of the sperm when they patients changed their mind regarding are frozen. Organelle defects may also have having more children, and financial some role in the loss of motility. constraints. Audrins et al. (1999) showed in In addition, reactive oxygen species can be a study that very low number of men formed during both freezing and thawing returned to use their stored sperm samples. processes, leading to decreased motility In this study 256 men who cryopreserved through peroxidation of the plasma lipid their semen before undergoing vasectomy, membrane. However, seminal plasma only 4 men returned to use them with the contains innate antioxidants. This provides a aim of achieving a pregnancy. The interval rationale for using neat semen during between storage and use ranged from 11 freezing (Smith et al, 1996). months to 10 years. And 258 men who cryopreserved their semen before D. Fertilization capacity chemotherapy and/or radiation therapy, There was no correlation reported between eighteen men returned to use their stored cancer type and outcomes such as semen. pregnancy or delivery rates (Hourvitz et al. 2008). Both viability and motility decrease The duration of storage since the diagnosis with cryopreservation in samples from both of their disease in the men who continued to healthy and ill individuals. Sperm store ranged from1 month to 16 years and morphology can change with the interval between storage and use of the cryopreservation, leading to lower motility, cryopreserved sperm ranged from 1 month and less potential for fertilization. to 7 years (Audrins et al. 1999). Fertilization capacity is impacted negatively because capacitation and the acrosome Art outcomes with banked semen reaction can be inhibited when specimens cryopreservation damages the membrane Cryopreservation of sperm provides a around the sperm head. Pentoxfylline readily available sperm and allows flexible treatment significantly increases the pre- coordination with female partner to undergo freezing sperm motility amplitude of the ART treatment cycle. While post-thaw lateral head displacement and the frequency semen quality is often not suitable for IUI, of spontaneous acrosome reactions however IVF-ICSI allows even the poorest (Schmidt et al. 2004). quality sperm to fertilize oocytes. Kelleher et al. (2001) reported 29 pregnancies in 64 This study found that the positive effects of men who underwent 85 ART cycles using pentoxifylline may be attributed to its frozen sperm (35 IUI, 28 IVF cycles, and 22 pentoxifylline intercepting ROS and causing IVF-ICSI cycles). The success rates of IVF increase of intracellular cAMP. and ICSI treatments using cryopreserved Cryopreservation of spermatozoa may have semen was comparable with fresh semen; decreased function due to reactive oxidative with average pregnancy rate of 54% ranging species, acrosomal dysfunction as well as from 33% and 73% (vanCasteren 2008). cellular changes that mark the spermatozoa Currently, there is limited data on ART for apoptosis. Pentoxifylline was found to

Utilization of spermatozoa for fertility preservation 162 treatment outcome of cryopreserved sperm motility during the freezing-thawing process from male cancer survivors (Tournaye et al. might still be viable and their fertilizing 2004). Only 18 of 258 patients who capacity might be preserved. This capacity cryopreserved their semen prior to may be better than the fertilizing capacity of chemotherapy used their frozen sperm for sperm cells that are primarily immotile when ART resulting in six pregnancies (Audrins et retrieved from the testes (Hauser et al. al. 1999). 2005) Challenges of sperm banking Hourvitz et a. (2008)l described the ART There are various ethical, legal and outcome in 118 male cancer survivors technical challenges associated with the undergoing 169 IVF-ICSI cycles, the largest banking of semen samples. Crawshaw, et series of couples treated with IVF-ICSI using al. (2004) has enumerated 5 challenges of cryopreserved sperm stored before cancer sperm banking dealing with young cancer therapy. They reported clinical pregnancy patients. These are: attributes of rate of 56.8%, which is comparable to the professionals, skills of professionals, average pregnancy rate achieved with other consent issues, issues relating to the effects male-factor patients in their center. of the process on the young men, and follow However, Lass et al. (1998) reported lower up services. This study also showed pregnancy rates. Only six out of 231 cancer difficulties in building and maintaining an patients with cryopreserved sperm returned adequate knowledge and skills base in this for infertility treatment after chemotherapy; field, and lack of appropriate training. two couples achieved pregnancy after IUI, Challenges also arise in regards to what to one couple after IVF, and two couples after be done with stored specimen in the event ICSI. Schmidt et al. (2004) reported a total of the patient’s death. Clear and precise of 151 ART cycles with clinical pregnancy instructions regarding the posthumous use rate of 14.8% after IUI and 38.6% following of stored gametes or gonadal tissue taken ICSI. from the patient along with informed consent should be recognized (Robertson 2005). A recent review on TESE studies described The health care policy makers are also a mean successful rate of 52% (Colpi et al. faced with challenges in allocating resources 2005). In cases associated with and making sperm banking accessible and cryptorchidism a significantly higher success affordable to people. rate than unexplained non-obstructive The sperm banks are faced with challenges azoospermia (NOA) have been reported of cross contamination of samples, as well (Raman and Schlegel 2003; Vernaeve et al. as providing efficient quality control. It is a 2004). Earlier studies comparing ICSI requirement for sperm banks to be outcomes in fresh vs. frozen-thawed cycles accredited by regulatory bodies and to be in in patients with NOA of all degrees of compliance with the current good tissue severity demonstrated that pregnancy rates practice regulations enforced by them. The were similar (Ben-Yosef et al. 1999). Hauser adherence to the good tissue practice et al. (2005) evaluated the outcome of fresh guidelines will prompt many of the and frozen TESE in the most difficult challenges of tissue banking. subgroup of NOA patients, those with very few, and sometimes exclusively immotile Conclusion sperm (severe hypospermatogenesis). Their Based on the etiological condition of the results indicated that pregnancies can be patients sperm can be collected by achieved at similar rates as with fresh ejaculation or by surgical retrieval from testicular sperm, even when motility is lost epididymis or testes. The sperm can be during the cryopreservation process. The used as fresh or frozen for latter use initial lack of motility correlated with a depending on the situation. Swim up and significant reduction in fertilization rates and density gradients are the two most widely with similar magnitude for both the fresh and used methods for sperm processing to frozen-thawed cycles; suggesting that post- separate motile sperm from seminal plasma, thaw loss of motility should be considered dead sperm and other cells. Sperm differently than primary lack of motility of cryopreservation is an important part of an fresh sperm. Motile sperm cells that lost infertility program for patients undergoing

Utilization of spermatozoa for fertility preservation 163 infertility treatments, fertility assurance for spermatozoa applying density-gradient vasectomy cases, and for fertility centrifugation: polysucrose media preservation in cancer patients before compared to Percoll media. J Assist starting chemotherapy or radiotherapy. Reprod Genet. 1997; 14(10):624-628. Arcidiacono A, Walt H, Campana A, Balerna Chemotherapy and radiation therapy or a M. The use of Percoll gradients for the combination of both has gonadotoxic effects preparation of subpopulations of human that lead to impairment of sperm quality spermatozoa. Int J Androl. 1983; resulting in infertility. Fertility preservation 6(5):433-445. options should be discussed with patients at Audrins P, Holden CA, McLachlan RI, an early stage of treatment planning for Kovacs GT. Semen storage for special cancer. The reported usage rates of patients purposes at Monash IVF from 1977 to coming back after cancer treatment and 1997. Fertil Steril. 1999; 72(1):179-181. utilising their cryopreserved samples are low Bagchi A, Woods EJ, Critser JK. varying between 5-10%. Although usage Cryopreservation and vitrification: recent rates are low, at least half of the couples can advances in fertility preservation benefit from getting pregnant with ART technologies. Expert Rev Med Devices. techniques utilising cryopreserved sperm. 2008; 5(3):359-370. Continuing research needs to focus on Bakos HW, Thompson JG, Feil D, Lane M. further improving cryopreservation protocols. Sperm DNA damage is associated with There is a need for guidance, training and assisted reproductive technology support, and availability of appropriate pregnancy. Int J Androl. 2008; 31(5):518- information system at national level for 526. different health and social care sectors that Ben-Yosef D, Yogev L, Hauser R, Yavetz H, are involved in treating cancer patients and Azem F, Yovel I, Lessing JB, Amit A. preserving their fertility by sperm banking. Testicular sperm retrieval and These sectors need to be well organized to cryopreservation prior to initiating ovarian face all of the challenges of sperm banking stimulation as the first line approach in services from diagnosis of the patient to patients with non-obstructive eventual discharge from the health system azoospermia. Hum Reprod. 1999; (Crawshaw et al. 2004). Fertility 14(7):1794-1801. preservation of younger cancer patients also Berger T, Marrs RP, Moyer DL. Comparison requires coordinated efforts and attention by of techniques for selection of motile oncologists and fertility specialists. They spermatozoa. Fertil Steril. 1985; have to be aware of all legal and ethical 43(2):268-273. issues and also what to do with posthumous Berthelsen JG. Sperm counts and serum stored specimen after the death of patient follicle-stimulating hormone levels before (Robertson 2005). and after radiotherapy and chemotherapy in men with testicular germ cell cancer. Reference Fertil Steril. 1984; 41(2):281-286. AbdelHafez F, Bedaiwy M, El-Nashar SA, Bolton VN, Braude PR. Preparation of Sabanegh E, Desai N. Techniques for human spermatozoa for in vitro cryopreservation of individual or small fertilization by isopycnic centrifugation on numbers of human spermatozoa: a self-generating density gradients. Arch systematic review. Hum Reprod Update. Androl. 1984; 13(2-3):167-176. 2009; 15(2):153-164. Bonetti TC, Pasqualotto FF, Queiroz P, Achille MA, Rosberger Z, Robitaille R, Lebel Iaconelli A, Jr., Borges E, Jr. Sperm S, Gouin JP, Bultz BD, Chan PT. banking for male cancer patients: social Facilitators and obstacles to sperm and semen profiles. Int Braz J Urol. banking in young men receiving 2009; 35(2):190-197; discussion 197- gonadotoxic chemotherapy for cancer: 198. the perspective of survivors and health Botchan A, Hauser R, Gamzu R, Yogev L, care professionals. Hum Reprod. 2006; Lessing JB, Paz G, Yavetz H. Sperm 21(12):3206-3216. quality in Hodgkin's disease versus non- Andersen CY, Grinsted J. A new method for Hodgkin's lymphoma. Hum Reprod. the purification of human motile 1997a; 12(1):73-76.

Utilization of spermatozoa for fertility preservation 164

Botchan A, Hauser R, Yogev L, Gamzu R, sperm banking to teenage males with Paz G, Lessing JB, Yavetz H. Testicular cancer. Hum Fertil (Camb) 7(1):23-30. cancer and spermatogenesis. Hum Crha I, Ventruba P, Zakova J, Huser M, Reprod. 1997b; 12(4):755-758. Kubesova B, Hudecek R, Jarkovsky J. Bunge RG, Sherman JK. Fertilizing capacity Survival and infertility treatment in male of frozen human spermatozoa. Nature. cancer patients after sperm banking. 1953; 172(4382):767-768. Fertil Steril. 2009; 91(6):2344-2348. Centola GM, Raubertas RF, Mattox JH. De Vos A, Nagy ZP, Van de Velde H, Joris Cryopreservation of human semen. H, Bocken G, Van Steirteghem A. Percoll Comparison of cryopreservatives, gradient centrifugation can be omitted in sources of variability, and prediction of sperm preparation for intracytoplasmic post-thaw survival. J Androl. 1992; sperm injection. Hum Reprod. 1997; 13(3):283-288. 12(9):1980-1984. Chapman RM, Sutcliffe SB, Malpas JS. de Vries MC, Bresters D, Engberts DP, Wit Male gonadal dysfunction in Hodgkin's JM, van Leeuwen E. Attitudes of disease. A prospective study. physicians and parents towards JAMA.1981; 245(13):1323-1328. discussing infertility risks and semen Cohen J, Garrisi GJ, Congedo-Ferrara TA, cryopreservation with male adolescents Kieck KA, Schimmel TW, Scott RT. diagnosed with cancer. Pediatr Blood Cryopreservation of single human Cancer. 2009; 53(3):386-391. spermatozoa. Hum Reprod. 19997; Devroey P, Liu J, Nagy Z, Goossens A, 12(5):994-1001. Tournaye H, Camus M, Van Steirteghem Colas G. Effect of initial freezing A, Silber S. Pregnancies after testicular temperature, addition of glycerol and sperm extraction and intracytoplasmic dilution on the survival and fertilizing sperm injection in non-obstructive ability of deep-frozen ram semen. J azoospermia. Hum Reprod. 1995; Reprod Fertil. 1975; 42(2):277-285. 10(6):1457-1460. Colpi GM, Piediferro G, Nerva F, Giacchetta Donoso P, Tournaye H, Devroey P. Which is D, Colpi EM, Piatti E. Sperm retrieval for the best sperm retrieval technique for intra-cytoplasmic sperm injection in non- non-obstructive azoospermia? A obstructive azoospermia. Minerva Urol systematic review. Hum Reprod Update. Nefrol. 2005; 57(2):99-107. 2007;13(6):539-549. Corona G, Jannini EA, Lotti F, Boddi V, De Eisenberg ML, Smith JF, Millstein SG, Vita G, Forti G, Lenzi A, Mannucci E, Walsh TJ, Breyer BN, Katz PP. Maggi M. Premature and delayed Perceived negative consequences of ejaculation: two ends of a single donor gametes from male and female continuum influenced by hormonal members of infertile couples. Fertil Steril. milieu. Int J Androl. 2010; Mar 19. [Epub 2009; Jun 10 [Epub ahead of print]. ahead of print] Esteves SC, Sharma RK, Thomas AJ, Jr., Craft I, Tsirigotis M. Simplified recovery, Agarwal A. Effect of swim-up sperm preparation and cryopreservation of washing and subsequent capacitation on testicular spermatozoa. Hum Reprod. acrosome status and functional 1995; 10(7):1623-1626. membrane integrity of normal sperm. Int Craft IL, Khalifa Y, Boulos A, Pelekanos M, J Fertil Womens Med. 2000a; 45(5):335- Foster C, Tsirigotis M. Factors 341. influencing the outcome of in-vitro Esteves SC, Sharma RK, Thomas AJ, Jr., fertilization with percutaneous aspirated Agarwal A. Improvement in motion epididymal spermatozoa and characteristics and acrosome status in intracytoplasmic sperm injection in cryopreserved human spermatozoa by azoospermic men. Hum Reprod. 1995; swim-up processing before freezing. 10(7):1791-1794. Hum Reprod. 2000b; 15(10):2173-2179. Crawshaw M, Glaser A, Hale J, Sloper P. Fasouliotis SJ, Safran A, Porat-Katz A, 2004. Professionals' views on the issues Simon A, Laufer N, Lewin A. A high and challenges arising from providing a predictive value of the first testicular fine fertility preservation service through needle aspiration in patients with non- obstructive azoospermia for sperm

Utilization of spermatozoa for fertility preservation 165

recovery at the subsequent attempt. Hum should we use fresh or frozen testicular Reprod. 2002; 17(1):139-142. spermatozoa? J Androl. 2005; 26(6):772- Fox RR. Preservation of rabbit spermatozoa: 778. fertility results from frozen semen. Proc Hauser R, Yogev L, Paz G, Yavetz H, Azem Soc Exp Biol Med. 1961; 108:663-665. F, Lessing JB, Botchan A. Comparison of Gellert-Mortimer ST, Clarke GN, Baker HW, efficacy of two techniques for testicular Hyne RV, Johnston WI. Evaluation of sperm retrieval in nonobstructive Nycodenz and Percoll density gradients azoospermia: multifocal testicular sperm for the selection of motile human extraction versus multifocal testicular spermatozoa. Fertil Steril. 1988; sperm aspiration. J Androl. 2006; 49(2):335-341. 27(1):28-33. Gil-Salom M, Minguez Y, Rubio C, De los Hobbie WL, Ogle SK, Ginsberg JP. Fertility Santos MJ, Remohi J, Pellicer A. Efficacy concerns for young males undergoing of intracytoplasmic sperm injection using cancer therapy. Semin Oncol Nurs. testicular spermatozoa. Hum Reprod. 2009; 25(4):245-250. 1995; 10(12):3166-3170. Hourvitz A, Goldschlag DE, Davis OK, Gilmore JA, Liu J, Gao DY, Critser JK. Gosden LV, Palermo GD, Rosenwaks Z. Determination of optimal cryoprotectants Intracytoplasmic sperm injection (ICSI) and procedures for their addition and using cryopreserved sperm from men removal from human spermatozoa. Hum with malignant neoplasm yields high Reprod. 1997; 12(1):112-118. pregnancy rates. Fertil Steril. 2008; Grunewald S, Paasch U, Glander HJ. 90(3):557-563. Enrichment of non-apoptotic human Howell SJ, Shalet SM. Testicular function spermatozoa after cryopreservation by following chemotherapy. Hum Reprod immunomagnetic cell sorting. Cell Tissue Update. 2001; 7(4):363-369. Bank. 2001; 2(3):127-133. Hsieh Y, Tsai H, Chang C, Lo H. Grunewald S, Paasch U, Said TM, Rasch M, Cryopreservation of human spermatozoa Agarwal A, Glander HJ. Magnetic- within human or mouse empty zona activated cell sorting before pellucidae. Fertil Steril. 2000; 73(4):694- cryopreservation preserves mitochondrial 698. integrity in human spermatozoa. Cell Isachenko E, Isachenko V, Katkov, II, Tissue Bank. 2006; 7(2):99-104. Dessole S, Nawroth F. Vitrification of Hallak J, Kolettis PN, Sekhon VS, Thomas mammalian spermatozoa in the absence AJ, Agarwal A. Sperm cryopreservation of cryoprotectants: from past practical in patients with testicular cancer. difficulties to present success. Reprod Urology. 1999a; 54(5):894-899. Biomed Online. 2003; 6(2):191-200. Hallak J, Kolettis PN, Sekhon VS, Thomas Isachenko V, Isachenko E, Katkov, II, AJ, Jr., Agarwal A. Cryopreservation of Montag M, Dessole S, Nawroth F, Van sperm from patients with leukemia: is it Der Ven H. Cryoprotectant-free worth the effort? Cancer. 1999b; cryopreservation of human spermatozoa 85(9):1973-1978. by vitrification and freezing in vapor: Hallak J, Sharma RK, Wellstead C, Agarwal effect on motility, DNA integrity, and A. Cryopreservation of human fertilization ability. Biol Reprod. 2004; spermatozoa: comparison of TEST-yolk 71(4):1167-1173. buffer and glycerol. Int J Fertil Womens Jurewicz J, Hanke W, Radwan M, Bonde Med. 2000; 45(1):38-42. JP. Environmental factors and semen Hauser R, Botchan A, Amit A, Ben Yosef D, quality. Int J Occup Med Environ Health. Gamzu R, Paz G, Lessing JB, Yogev L, 2009; 22(4):305-329. Yavetz H. Multiple testicular sampling in Kelleher S, Wishart SM, Liu PY, Turner L, Di non-obstructive azoospermia--is it Pierro I, Conway AJ, Handelsman DJ. necessary? Hum Reprod. 1998; Long-term outcomes of elective human 13(11):3081-3085. sperm cryostorage. Hum Reprod. 2001; Hauser R, Yogev L, Amit A, Yavetz H, 16(12):2632-2639. Botchan A, Azem F, Lessing JB, Ben- Kobayashi H, Larson K, Sharma RK, Nelson Yosef D. Severe hypospermatogenesis DR, Evenson DP, Toma H, Thomas AJ, in cases of nonobstructive azoospermia: Agarwal A. DNA damage in patients with

Utilization of spermatozoa for fertility preservation 166

untreated cancer as measured by the University Hospital. Hum Reprod. sperm chromatin structure assay. Fertil 2009a; 24(1):37-44. Steril. 2001; 75(3):469-475. Menon U, Gentry-Maharaj A, Hallett R, Ryan Lacerda HM, Akre O, Merletti F, Richiardi L. A, Burnell M, Sharma A, Lewis S, Davies Time trends in the incidence of testicular S, Philpott S, Lopes A, Godfrey K, Oram cancer in childhood and young D, Herod J, Williamson K, Seif MW, Scott adulthood. Cancer Epidemiol Biomarkers I, Mould T, Woolas R, Murdoch J, Dobbs Prev. 2009; 18(7):2042-2045. S, Amso NN, Leeson S, Cruickshank D, Lass A, Akagbosu F, Abusheikha N, McGuire A, Campbell S, Fallowfield L, Hassouneh M, Blayney M, Avery S, Singh N, Dawnay A, Skates SJ, Parmar Brinsden P. A programme of semen M, Jacobs I. Sensitivity and specificity of cryopreservation for patients with multimodal and ultrasound screening for malignant disease in a tertiary infertility ovarian cancer, and stage distribution of centre: lessons from 8 years' experience. detected cancers: results of the Hum Reprod. 1998; 13(11):3256-3261. prevalence screen of the UK Lass A, Akagbosu F, Brinsden P. Sperm Collaborative Trial of Ovarian Cancer banking and assisted reproduction Screening (UKCTOCS). Lancet Oncol. treatment for couples following cancer 2009b;10(4):327-340. treatment of the male partner. Hum Mori M. A secular perspective on 21st Reprod Update. 2001; 7(4):370-377. century ethics in human reproduction: Levin HS. Testicular biopsy in the study of why religious views and attitudes are male infertility: its current usefulness, becoming obsolete and possibly histologic techniques, and prospects for dangerous. Reprod Biomed Online. the future. Hum Pathol. 1979; 10(5):569- 2008; 17 (Suppl 3):49-51. 584. Mortimer D. Sperm recovery techniques to Lewin A, Weiss DB, Friedler S, Ben-Shachar maximize fertilizing capacity. Reprod I, Porat-Katz A, Meirow D, Schenker JG, Fertil Dev. 1994; 6(1):25-31. Safran A. Delivery following Mulhall JP, Ghaly SW, Aviv N, Ahmed A. intracytoplasmic injection of mature The utility of optical loupe magnification sperm cells recovered by testicular fine for testis sperm extraction in men with needle aspiration in a case of nonobstructive azoospermia. J Androl. hypergonadotropic azoospermia due to 2005; 26(2):178-181. maturation arrest. Hum Reprod. 1996; Nawroth F, Isachenko V, Dessole S, Rahimi 11(4):769-771. G, Farina M, Vargiu N, Mallmann P, Mahadevan M, Trounson AO. Effect of Dattena M, Capobianco G, Peters D, cryoprotective media and dilution Orth I, Isachenko E. Vitrification of methods on the preservation of human human spermatozoa without spermatozoa. Andrologia. 1983; cryoprotectants. Cryo Letters. 2002; 15(4):355-366. 23(2):93-102. Mazur P, Rall WF, Rigopoulos N. Relative Nijs M, Vanderzwalmen P, Vandamme B, contributions of the fraction of unfrozen Segal-Bertin G, Lejeune B, Segal L, van water and of salt concentration to the Roosendaal E, Schoysman R. Fertilizing survival of slowly frozen human ability of immotile spermatozoa after erythrocytes. Biophys J. 1981; 36(3):653- intracytoplasmic sperm injection. Hum 675. Reprod. 1996; 11(10):2180-2185. Meirow D, Schenker JG. The current status Nishikawa Y. Studies on the preservation of of sperm donation in assisted raw and frozen horse semen. J Reprod reproduction technology: ethical and Fertil. 1975; Suppl(23):99-104. legal considerations. J Assist Reprod Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Genet. 1997; 14(3):133-138. Karahuseyinoglu S. Effects of Menon S, Rives N, Mousset-Simeon N, cryopreservation on sperm parameters Sibert L, Vannier JP, Mazurier S, Masse and ultrastructural morphology of human L, Duchesne V, Mace B. Fertility spermatozoa. J Assist Reprod Genet. preservation in adolescent males: 2008; 25(8):403-411. experience over 22 years at Rouen Padron OF, Brackett NL, Sharma RK, Lynne CM, Thomas AJ, Agarwal A. Seminal

Utilization of spermatozoa for fertility preservation 167

reactive oxygen species and sperm molecular sperm preparation technique. motility and morphology in men with J Androl. 2008; 29(2):134-142. spinal cord injury. Fertil Steril. 1997; Said TM, Grunewald S, Paasch U, Rasch M, 67(6):1115-1120. Agarwal A, Glander HJ. Effects of Palermo G, Joris H, Devroey P, Van magnetic-activated cell sorting on sperm Steirteghem AC. Pregnancies after motility and cryosurvival rates. Fertil intracytoplasmic injection of single Steril. 2005; 83(5):1442-1446. into an oocyte. Lancet. Salonia A, Gallina A, Matloob R, Rocchini L, 1992; 340(8810):17-18. Sacca A, Abdollah F, Colombo R, Suardi Polge C. Fertilizing capacity of bull N, Briganti A, Guazzoni G, Rigatti P, spermatozoa after freezing at 79 degrees Montorsi F. Is sperm banking of interest C. Nature. 1952; 169(4302):626-627. to patients with nongerm cell urological Pousette A, Akerlof E, Rosenborg L, cancer before potentially fertility Fredricsson B. Increase in progressive damaging treatments? J Urol. 2009; motility and improved morphology of 182(3):1101-1107. human spermatozoa following their Schlegel PN. Testicular sperm extraction: migration through Percoll gradients. Int J microdissection improves sperm yield Androl1986; 9(1):1-13. with minimal tissue excision. Hum Prins GS, Weidel L. A comparative study of Reprod. 1999; 14(1):131-135. buffer systems as cryoprotectants for Schlegel PN, Berkeley AS, Goldstein M, human spermatozoa. Fertil Steril. 1986; Cohen J, Alikani M, Adler A, Gilbert BR, 46(1):147-149. Rosenwaks Z. Epididymal micropuncture Pursel VG, Johnson LA. Freezing of boar with in vitro fertilization and oocyte spermatozoa: fertilizing capacity with micromanipulation for the treatment of concentrated semen and a new thawing unreconstructable obstructive procedure. J Anim Sci. 1975; 40(1):99- azoospermia. Fertil Steril. 1994; 102. 61(5):895-901. Quinn GP, Vadaparampil ST, Lee JH, Schmidt KL, Larsen E, Bangsboll S, Jacobsen PB, Bepler G, Lancaster J, Meinertz H, Carlsen E, Andersen AN. Keefe DL, Albrecht TL. Physician referral Assisted reproduction in male cancer for fertility preservation in oncology survivors: fertility treatment and outcome patients: a national study of practice in 67 couples. Hum Reprod. 2004; behaviors. J Clin Oncol. 2009; 19(12):2806-2810. 27(35):5952-5957. Schover LR. Psychosocial aspects of Raman JD, Schlegel PN. Testicular sperm infertility and decisions about extraction with intracytoplasmic sperm reproduction in young cancer survivors: a injection is successful for the treatment of review. Med Pediatr Oncol. 1999; nonobstructive azoospermia associated 33(1):53-59. with cryptorchidism. J Urol. 2003; 170(4 Schover LR, Brey K, Lichtin A, Lipshultz LI, Pt 1):1287-1290. Jeha S. Oncologists' attitudes and Robertson JA. Cancer and fertility: ethical practices regarding banking sperm and legal challenges. J Natl Cancer Inst before cancer treatment. J Clin Oncol. Monogr. 2005; 2005 (34):104-106. 2002; 20(7):1890-1897. Romero J, Remohi J, Minguez Y, Rubio C, Schrader M, Muller M, Straub B, Miller K. Pellicer A, Gil-Salom M. Fertilization after The impact of chemotherapy on male intracytoplasmic sperm injection with fertility: a survey of the biologic basis and cryopreserved testicular spermatozoa. clinical aspects. Reprod Toxicol. 2001; Fertil Steril. 1996; 65(4):877-879. 15(6):611-617. Russell LD, Rogers BJ. Improvement in the Scott L, Smith S. Mouse in vitro fertilization, quality and fertilization potential of a embryo development and viability, and human sperm population using the rise human sperm motility in substances used technique. J Androl. 1987; 8(1):25-33. for human sperm preparation for assisted Said TM, Agarwal A, Zborowski M, reproduction. Fertil Steril. 1997; Grunewald S, Glander HJ, Paasch U. 67(2):372-381. Utility of magnetic cell separation as a

Utilization of spermatozoa for fertility preservation 168

Seager SW, Fletcher WS. Progress on the patients. Fertility and Sterility. 2008; use of frozen semen in the dog. Vet Rec. 90(6):2245-2250. 1973; 92(1):6-10. Vernaeve V, Krikilion A, Verheyen G, Van Serafini P, Blank W, Tran C, Mansourian M, Steirteghem A, Devroey P, Tournaye H. Tan T, Batzofin J. Enhanced penetration Outcome of testicular sperm recovery of zona-free hamster ova by sperm and ICSI in patients with non-obstructive prepared by Nycodenz and Percoll azoospermia with a history of gradient centrifugation. Fertil Steril. orchidopexy. Hum Reprod. 2004; 1990; 53(3):551-555. 19(10):2307-2312. Smith R, Vantman D, Ponce J, Escobar J, Williams DH, Karpman E, Sander JC, and Lissi E. (1996) Total antioxidant Spiess PE, Pisters LL, Lipshultz LI. capacity of human seminal plasma. Pretreatment semen parameters in men Hum Reprod. 1996; 11(8):1655–1660. with cancer. J Urol. 2009; 181(2):736- Strehler E, Baccetti B, Sterzik K, Capitani S, 740. Collodel G, De Santo M, Gambera L, Yildiz C, Ottaviani P, Law N, Ayearst R, Liu Piomboni P. Detrimental effects of L, McKerlie C. Effects of polyvinylpyrrolidone on the ultrastructure cryopreservation on sperm quality, of spermatozoa (Notulae seminologicae nuclear DNA integrity, in vitro fertilization, 13). Hum Reprod. 1998; 13(1):120-123. and in vitro embryo development in the Thomson LK, Fleming SD, Aitken RJ, De mouse. Reproduction. 2007; 133(3):585- Iuliis GN, Zieschang JA, Clark AM. 595. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009; 24(9):2061-2070. Tournaye H, Devroey P, Liu J, Nagy Z, Lissens W, Van Steirteghem A. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil Steril. 1994; 61(6):1045-1051. Tournaye H, Goossens E, Verheyen G, Frederickx V, De Block G, Devroey P, Van Steirteghem A. Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects. Hum Reprod Update. 2004; 10(6):525-532. Tschudin S, Bitzer J. Psychological aspects of fertility preservation in men and women affected by cancer and other life- threatening diseases. Hum Reprod Update. 2009; 15(5):587-597. Turek PJ, Givens CR, Schriock ED, Meng MV, Pedersen RA, Conaghan J. Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine- needle aspiration mapping in patients with nonobstructive azoospermia. Fertil Steril. 1999; 71(3):552-557. vanCasteren NJ. Use rate and assisted reproduction technologies outcome of cryopreserved semen from 629 cancer

Top View