9.4.15 Cryopreservation of sperm

Malignant diseases in adolescence and younger adults such as testicular cancer, lymphomas and leukaemia have long-term survival rates of up to 80% if treated adequately. As a result, long-term quality of life, including reproductive health, has become increasingly important. The cryopreservation of sperm from oncological patients represents the most frequent indication for the procedure. Depending on the substance and dosages administered, chemo- and/or radiotherapy, as well as surgical intervention, can lead to persistent azoospermia independent of the patienńs pubertal status. Theoretically, hormonal gonadal protection and retransplantation of germ cell stem cells preserved prior to chemotherapy offer options to preserve fertility, but neither approach has yet proven to be of clinical benefit. Therefore at present, cryopreservation of sperm prior to oncological therapy offers the only possibility of circumventing the deleterious effects of disease and therapy on fertility, thereby contributing to the personal stabilization of the predominantly young patients in this critical situation.

Currently, men undergoing diagnostic and therapeutic testicular biopsies, performed to detect sperm possibly remaining in the testis for use in intracytoplasmic sperm injection (ICSI) (Chapter 9.4.14), may opt for cryopreservation. Until histological examination is complete, the remaining tissue remains frozen, for later use or subsequent thawing or disposal.

The first observation that the motility of human spermatozoa can be preserved after freezing and thawing was made by Lazzaro Spallanzani in 1776. However, feasible cryopreservation, with acceptable survival rates of spermatozoa, was established only after the introduction of cryoprotective substances and storage of the samples in liquid nitrogen in the second half of the 20th century. After the advent of ICSI, subsequent procedures for the cryopreservation of testicular tissue with later sperm extraction (testicular sperm extraction, TESE) and microsurgical epididymal sperm aspiration (MESA) were established at the end of the 20th century.

Most patients deciding in favour of cryopreservation of semen are those with malignant diseases (Table 9.4.15.1). These diseases often strike when family planning has not started or is not yet finished; only a minority of patients had already fathered a child before they became ill (14% in our centre).

In younger adults with testicular tumours or other malignancies (e.g. Hodgkin’s disease, leukaemia, bone cancer), impairment of spermatogenesis due to the disease itself is known to occur (Fig. 9.4.15.1), although the underlying causes are not fully understood (see 9.4.7). In addition to the reduced baseline semen parameters of oncological patients (only 17% of our adults and 23% of our adolescents showed normozoospermia as defined by WHO criteria (1)), sperm motility is further impaired to around 50% of baseline values by the cryopreservation process itself. However, since the advent of ICSI, reduced sperm concentrations and motility do not play the most important role in predicting the success of assisted fertilization techniques (Chapter 9.4.14), and cryopreservation can be offered successfully to most patients (89% of our oncological patients).

Cryopreservation can be offered not only to adults but also to adolescents with malignancies (our youngest patient was 13.5 years). As semen parameters prior to and after cryopreservation in 14 to 17 year-old boys are comparable to those from adults, cryopreservation of sperm should be considered when counselling adolescents and their parents prior to toxic treatments (1). The most predictive parameter for successful cryopreservation of sperms in the adolescents appears to be testicular volume, which should be normally developed for age and should show signs of spermarche as evidenced by a unilateral testicular volume above 5 ml (1).

As vasectomy is an invasive, potentially irreversible contraceptive method, cryopreservation of sperm may be considered for these patients. Generally 2–7% of vasectomized patients later request reversal. Cryopreserved semen obtained prior to vasectomy or aspirated during later microsurgical vasovasostomy or vasoepididymostomy may offer the chance to father a child by assisted reproduction techniques in cases of unsuccessful or impossible restoration of fertility. Cryopreservation of sperm during vasoepididymostomy is especially important, because of a reported 35% rate of azoospermia after microsurgical vasoepididymostomy.

Only 14% of men with spinal cord injuries (average age at injury <30 years) reported ejaculations and only 1.8% were able to achieve a pregnancy with their spouse. By electrovibration stimulation or rectal electric stimulation ejaculations may be achieved and semen may be successfully used for assisted reproduction techniques. As repeated electrostimulation is inconvenient for the patient, cryopreservation of ejaculated sperm after electrostimulation or after TESE offers the advantage of storing semen samples for later use in assisted reproduction techniques.

MESA allows collection of semen specimens from patients with postinfectious obstructive azoospermia, congenital bilateral aplasia of the vas deferens (CBAVD), or abnormalities of the epididymides that cannot be successfully treated by reconstructive microsurgery. Aspirated semen not used for artificial reproduction techniques can be cryopreserved and used for further treatment cycles in assisted reproduction. As fertilization rates were not different with the easier to perform TESE, nowadays MESA is less important.

Testicular sperm from patients with severe impairment of semen parameters (cryptozoospermia or azoospermia), due to focal Sertoli cell-only (SCO) syndrome or incomplete arrest of spermatogenesis, and testicular sperm with obstructive azoospermia (e.g. due to infections) may be used after TESE in combination with ICSI (Chapter 9.4.14). Overall, spermatozoa may be extracted from testicular tissue in up to 70% of azoospermic men. Pregnancy rates with testicular sperm vary depending on the centre and, especially, depending on the severity of spermatogenic impairment, but usually fall between 10 and 30%.

Cryopreservation of semen from healthy donors for heterologous assisted fertilization is practiced in various countries and depends on applicable laws. As semen quality of donors is generally much better than in male infertility patients, much of the donor semen is used for intrauterine insemination (Chapter 9.4.14). However, where legal regulations allow, donor spermatozoa may be used also for IVF or ICSI if necessary.

Finally, cryopreservation of sperm plays an increasing role in establishing internal and external quality control systems in andrology.

On average, 2 to 3 semen samples per patient are frozen. Prior to cryopreservation the liquefied ejaculate is analysed according to WHO guidelines (see 9.3.3). Mainly during the process of thawing, the number of live, membrane-intact sperm gradually decreases due to intracellular formation of ice crystals and increasing concentrations of surrounding solutes (2). Cryoprotective media based on a mixture of penetrating (e.g. glycerine, sorbitol, propanediol) and non-penetrating (e.g. glucose, human serum albumin, egg yolk) ingredients prevent cell damage by reducing intracellular ice crystal formation (penetrating) or stabilising the cell membrane (non-penetrating). In addition, nearly all cryoprotective media contain a HEPES pH buffer and antibiotics based on a culture media.

After liquefaction the ejaculate is carefully mixed with the same volume of a cryoprotective medium. The diluted sample is filled into straws, which offer optimal temperature distribution during cooling. These straws are then frozen, mostly using a computerized, automated system guaranteeing standardized cryopreservation conditions. There are no uniform guidelines available for the optimal freezing time, but a freezing velocity of 8–21°C per minute is used by most groups. After −196°C is reached, the semen samples are stored in liquid nitrogen, ideally in the vapour phase. Thawing velocity should be adapted to freezing velocity using a programmed cryo-machine, room temperature, or a water bath warmed to 37 °C. Following cryopreservation, immediate thawing and analysis of a small aliquot of the sample is helpful for counselling the patient regarding possible later use of his cryopreserved sperm.

No systematic or prospective studies have investigated the influence of modern standardised cryopreservation techniques on sperm quality during long-term storage. Published data on pregnancies conceived after artificial reproduction techniques with cryopreserved sperm of oncological patients suggest that current methodology allows frozen samples to survive for at least 30 years (3).

For cryopreservation and subsequent storage of human spermatozoa it is necessary to comply with local recommendations and legislation regarding coding and traceability of samples, documentation of procedures, staff expertise and equipment, an adverse events/serious adverse events reporting system, ensuring a constant liquid nitrogen supply, as well as genetic and infection screening (see guidelines in American Fertility Society; British Andrology Society; Canadian Fertility and Andrology Society; Fertility Society of Australia; EU Directive 2004/23/EC).

Systematic studies and sufficient data regarding the number of artificial reproduction attempts performed with cryopreserved sperm are not available. In a review paper (3) covering the period between 1983 and 1992 (i.e. before the advent of ICSI), a total of 117 pregnancies and 115 births after insemination or in vitro fertilization (IVF) with the cryopreserved semen samples of oncological patients were reported. However, as sperm motility was regarded as one of the important parameters for success rates in insemination and IVF therapies, and as sperm motility is generally lower in cryopreserved specimens, fertilization rates and pregnancy rates per patient were indeed considerably lower with cryopreserved semen than with fresh semen, when using conventional artificial reproduction techniques. Therefore, today ICSI should be considered as a first choice when using cryopreserved sperm.

Only small cohort studies have been published concerning the efficacy of ICSI with the use of cryopreserved sperm. However, in the German IVF register (DIR) from 1997 to 2007, a total of 4457 embryo transfers were performed after ICSI with cryopreserved ejaculated sperm (4). The overall pregnancy rate per embryo transfer was 26%, which is comparable with the pregnancy rate with sperm used for ICSI after fresh orthograde ejaculation (28%). For TESE the DIR registered 17527 embryo transfers after ICSI with fresh and cryopreserved TESE samples from 1995 to 2007. The overall pregnancy rate per embryo transfer was 23%.

Concern has been raised about an increased genetic risk for the offspring of patients suffering from malignancies, arising from the malignant disease itself or from the use of ICSI with their cryopreserved sperm. Documented clinical data from IVF centres and data obtained from the offspring of oncological patients both indicate that no increased genetic risk and no increased risk for malformation exist, arising from the underlying oncological disease or the applied assisted fertilization technique with cryopreserved spermatozoa.

Efficient cryopreservation of small numbers of sperm may reduce the number of surgical interventions, thus avoiding complications and expenses associated with repeated surgery. A new technique is the cryopreservation of single human sperm. A recent systematic review identified 30 small cohort studies, in which seven types of nonbiological sperm storage systems, or carriers, and two biological carriers were used (5). The recovery rate of spermatozoa in these studies was 79.5% with a range of 59–100% with motility rates starting from 0 to 100% (survival rate range 8–85%). In studies that attempted fertilization, the overall average fertilization rate was 42.5% with a range of 18–67%. In studies where sperm were used for ICSI (13 patients, 12 cycles) four pregnancies were reported. However, despite first promising results, no consensus has been found to date regarding the ideal carrier for cryopreservation of small numbers of spermatozoa for clinical purposes, in terms of efficacy and compliance with local recommendations and legislations.