Sperm Cryopreservation Prior to Gonadotoxic Treatment

Experience of a Single Academic Centre Over 4 Decades

Nandini Shankara-Narayana; Irene Di Pierro; Carolyn Fennell; Lam P. Ly; Fay Bacha; Ljubica Vrga; Sasha Savkovic; Leo Turner; Veena Jayadev; Ann J. Conway; David J. Handelsman


Hum Reprod. 2019;34(5):795-803. 

In This Article

Materials and Methods

Sperm Cryostorage Program

Since 1978 sperm cryostorage was offered to men referred to Andrology Department, Concord Repatriation General Hospital, Sydney, NSW (prior to 1999 as the Andrology Unit, Royal Prince Alfred Hospital) in large urban tertiary referral teaching hospitals of the University of Sydney Medical School. The sperm cryostorage program operated within the national health scheme (Medicare) as a NSW public hospital service provided without charge to eligible patients with a proven ongoing need for cryostorage due to impending iatrogenic gamete loss. Any sufficiently mature male adolescent or adult, without age limits, who could collect semen samples by masturbation was eligible for sperm cryostorage. Referrals came from medical or radiation oncologists, surgeons or other specialists for men who (i) had not completed their family and (ii) were scheduled to undergo gonadotoxic treatment risking gamete loss. Cryostorage was not offered where gamete loss was not expected (e.g. vasectomy) or where impaired fertility could be rectified by other medical or surgical treatment (e.g. gonadotrophin deficiency, neurogenic erectile failure).

The signed consent form included an obligation for annual follow-up review after completion of treatment to verify an ongoing need for cryostorage. Those wishing to allow post-mortem use of their sperm to induce pregnancy could nominate their current female conjugal partner. That nomination could be updated at follow-up visits by adding, deleting or changing the specified partner. All men underwent initial and follow-up review including a general and reproductive history (cryptorchidism or testicular pathology, marital and fertility status, smoking and alcohol use, recent fever or weight loss) and physical examination including estimating testicular volume by orchidometry and androgenization. Fertility counselling was provided for their disease and its treatment including contraception advice and future use of stored sperm. The patients were also counselled about follow-up required to establish ongoing need for cryostorage without out-of-pocket cost or else disposal of samples by discarding as no longer required or after prolonged failure of follow-up or by transfer to a private facility.

Annual follow-up reviewed the need for ongoing cryostorage based on history of recent paternity, recovery of sperm output together with testicular examination, semen analysis and measurement of reproductive hormones (serum FSH, LH and testosterone). Where fertility or sperm output had returned and further gonadotoxic treatment was not anticipated, cryostored sperm samples were either discarded or transferred to a private cryostorage facility. Follow-up was organized by an experienced cryostorage nurse contacting the man, his family, treating doctors, general practitioner or treating specialists using phone, Internet and mail as well as searching relevant databases including the death registry. If this failed to secure a follow-up visit, a case conference review considered the man's disease, its treatment and likely recovery of fertility and may authorize a 'sunset letter' sent by registered post indicating an intention with 3 months' notice to discard sperm.

Semen Analysis and Sperm Cryopreservation

Semen was produced by masturbation in a collecting room near the Clinical Andrology laboratory. An abstinence interval of 2–3 days was recommended but sperm was stored regardless of the recorded abstinence interval. Men were encouraged to provide three (and up to six) semen collections on alternate days prior to commencing gonadotoxic therapy with any viable sperm cryostored. Cryostorage was also undertaken after starting gonadotoxic treatment with affected men advised of hypothetical risks of mutational effects on sperm, issues that could be reviewed later prior to elective use of stored sperm. Neither electro-ejaculation nor surgery (testicular biopsy or IVF-related sperm extraction procedures) were used to collect sperm.

Semen samples were processed after liquefaction according to the contemporaneous methods as specified by the WHO Laboratory Manual for the Examination and Processing of Human Semen, first to fifth editions. The semen analysis complied with all aspects of Björndahl's checklist (Björndahl et al., 2016) except for dilutions which were compliant with the WHO manual. Abstinence interval, semen volume, sperm concentration, morphology, motility and vitality were recorded. For cryostorage, semen was diluted 1:1 (v/v) with sperm freeze medium and 0.5 ml aliquots were transferred in to plastic straws for freezing in graduated vapour phase nitrogen over 30 min before transfer to larger vapour phase nitrogen tanks for storage at −185°C.


Semen samples were analysed for semen volume and sperm concentration from the first semen sample or a median value of these parameters where more than one sample was available. Total sperm output was calculated from the semen volume and sperm concentration. Sperm data were analysed after cube-root transformation to normalize distribution (Handelsman, 2002) with back-transformed results to the natural scale for clarity and as an estimate of the median. Correlations by rank and parametric methods gave near identical results for this large sample size. Data were analysed according to time in storage, diagnostic category, marital status, alcohol intake, smoking, prior fertility or infertility or known testicular pathology, cryptorchidism, history of recent fever or weight loss by survival analysis, analysis of variance and t-test as appropriate using SPSS 21 and NCSS v12 software. Differences between first and median sperm variables were analysed without data transformation consistent with the Central Limit Theorem and confirmed by Box-Cox analysis. Within the state of New South Wales, the remoteness or regionality (relative to major cities) of a patient's address was assessed based on postcodes using the Accessibility/Remoteness Index of Australia (Information and Research Branch, 2001) and expected prevalence of cancer in age-matched men was based on data from NSW Cancer Registry data.