Purpose To develop guidance to practicing oncologists about available fertility preservation methods and related issues in people treated for cancer. Methods An expert panel and a writing committee were formed. The questions to be addressed by the guideline were determined, and a systematic review of the literature from 1987 to 2005 was performed, and included a search of online databases and consultation with content experts. Results The literature review found many cohort studies, case series, and case reports, but relatively few randomized or definitive trials examining the success and impact of fertility preservation methods in people with cancer. Fertility preservation methods are used infrequently in people with cancer. Recommendations As part of education and informed consent before cancer therapy, oncologists should address the possibility of infertility with patients treated during their reproductive years and be prepared to discuss possible fertility preservation options or refer appropriate and interested patients to reproductive specialists. Clinician judgment should be employed in the timing of raising this issue, but discussion at the earliest possible opportunity is encouraged. Sperm and embryo cryopreservation are considered standard practice and are widely available; other available fertility preservation methods should be considered investigational and be performed in centers with the necessary expertise.

https://ascopubs.org/doi/pdf/10.1200/jco.2006.06.5888

American Society of Clinical Oncology Recommendations on Fertility Preservation in Cancer Patients

Disorders of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of Sertoli cells. Failure of Sertoli cells to mature, with consequent inability to express functions capable of supporting spermatogenesis, is a prime example. In a similar way, failure of Sertoli cells to proliferate normally at the appropriate period in life will result in reduced production of spermatozoa in adulthood. This review focuses on the control of proliferation of Sertoli cells and functional maturation, and is motivated by concerns about 'testicular dysgenesis syndrome' in humans, a collection of common disorders (testicular germ-cell cancer, cryptorchidism, hypospadias and low sperm counts) which are hypothesized to have a common origin in fetal life and to reflect abnormal function of Sertoli (and Leydig) cells. The timing of proliferation of Sertoli cells in different species is reviewed, and the factors that govern the conversion of an immature, proliferating Sertoli cell to a mature, non-proliferating cell are discussed. Protein markers of maturity and immaturity of Sertoli cells in various species are reviewed and their usefulness in studies of human testicular pathology are discussed. These markers include anti-Mullerian hormone, aromatase, cytokeratin-18, GATA-1, laminin alpha5, M2A antigen, p27(kip1), sulphated glycoprotein 2, androgen receptor and Wilms' tumour gene. A scheme is presented for characterization of Sertoli-cell only tubules in the adult testis according to whether or not there is inherent failure of maturation of Sertoli cells or in which the Sertoli cells have matured but there is absence, or acquired loss, of germ cells. Functional 'de-differentiation' of Sertoli cells is considered. It is concluded that there is considerable evidence to indicate that disorders of maturation of Sertoli cells may be a common underlying cause of human male reproductive disorders that manifest at various life stages. This recognition emphasizes the important role that animal models must play to enable identification of the mechanisms via which failure of proliferation and maturation of Sertoli cells can arise, as this failure probably occurs in fetal life.

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Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood

Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.

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The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

Background The disorders comprising human 'testicular dysgenesis syndrome' (TDS) may be increasing in incidence. TDS originates in fetal life but the mechanisms are not known, and discerning them requires an animal model. Methods and results The study investigated whether male rats exposed in utero to dibutyl phthalate [DBP; 500 mg/kg on gestational days (GD) 13-21] would provide a suitable model for human TDS. DBP induced a high rate (>60%) of cryptorchidism (mainly unilateral), hypospadias, infertility and testis abnormalities, similar to those in human TDS. Cell-specific immunohistochemistry and confocal microscopy were used to track development of Sertoli [anti-Mullerian hormone (AMH), Wilm's tumour (WT-1) protein, p27(kip)], Leydig [3beta-hydroxysteroid dehydrogenase (3beta-HSD)], germ (DAZL protein) and peritubular myoid (smooth muscle actin) cells from fetal life to adulthood. In scrotal and cryptorchid testes of DBP-exposed males, areas of focal dysgenesis were found that contained Sertoli and Leydig cells, and gonocytes and partially formed testicular cords; these dysgenetic areas were associated with Leydig cell hyperplasia at all ages. Suppression ( approximately 90%) of testicular testosterone levels on GD 19 in DBP-exposed males, coincident with delayed peritubular myoid cell differentiation, may have contributed to the dysgenesis. Double immunohistochemistry using WT-1 (expressed in all Sertoli cells) and p27(kip) (expressed only in mature Sertoli cells) revealed immature Sertoli cells in dysgenetic areas. DBP-exposed animals also exhibited Sertoli cell-only (SCO) tubules, sporadically in scrotal and predominantly in cryptorchid, testes, or foci of SCO within normal tubules in scrotal testes. In all SCO areas the Sertoli cells were immature. Intratubular Leydig cells were evident in DBP-exposed animals and, where these occurred, Sertoli cells were immature and spermatogenesis was absent. Abnormal Sertoli cell-gonocyte interaction was evident at GD 19 in DBP-exposed rats coincident with appearance of multinucleated gonocytes, although these disappeared by postnatal day 10 during widespread loss of germ cells. Conclusions Abnormal development of Sertoli cells, leading to abnormalities in other cell types, is our hypothesized explanation for the abnormal changes in DBP-exposed animals. As the testicular and other changes in DBP-exposed rats have all been reported in human TDS, DBP exposure in utero may provide a useful model for defining the cellular pathways in TDS.

Human ‘testicular dysgenesis syndrome’: a possible model using in‐utero exposure of the rat to dibutyl phthalate

Male infertility is a multifactorial pathological condition affecting approximately 7% of the male population. The genetic landscape of male infertility is highly complex as semen and testis histological phenotypes are extremely heterogeneous, and at least 2,000 genes are involved in spermatogenesis. The highest frequency of known genetic factors contributing to male infertility (25%) is in azoospermia, but the number of identified genetic anomalies in other semen and aetiological categories is constantly growing. Genetic screening is relevant for its diagnostic value, clinical decision making, and appropriate genetic counselling. Anomalies in sex chromosomes have major roles in severe spermatogenic impairment. Autosome-linked gene mutations are mainly involved in central hypogonadism, monomorphic teratozoospermia or asthenozoospermia, congenital obstructive azoospermia, and familial cases of quantitative spermatogenic disturbances. Results from whole-genome association studies suggest a marginal role for common variants as causative factors; however, some of these variants can be important for pharmacogenetic purposes. Results of studies on copy number variations (CNVs) demonstrate a considerably higher CNV load in infertile patients than in normozoospermic men, whereas whole-exome analysis has proved to be a highly successful diagnostic tool in familial cases of male infertility. Despite such efforts, the aetiology of infertility remains unknown in about 40% of patients, and the discovery of novel genetic factors in idiopathic infertility is a major challenge for the field of androgenetics. Large, international, and consortium-based whole-exome and whole-genome studies are the most promising approach for the discovery of the missing genetic aetiology of idiopathic male infertility.

Genetics of male infertility.

Recently, microdeletions in the azoospermic factor region ofthe Y chromosome, in addition to chromosomal anomalies,have been detected in men with azoospermia or severeoligozoospermia. In this study we evaluated the molecularand cytogenetic defects of infertile men. The frequency of Ymicrodeletions among 105 azoospermic, 28 oligozoospermicand 32 fertile men was tested on lymphocyte DNA using aseries of 20 sequence-tagged sites. In addition, microdele-tions were evaluated on testicular-derived DNA among 26azoospermic patients who underwent testicular biopsy andin whom no sperm cells could be identiﬁed. Karyotypeanalysis was performed on 72 of the infertile patients.Deletions were detected in 6.7% azoospermic and 3.6%oligozoospermic men. No deletions were identiﬁed amongthe fertile men. Identical results were obtained with DNAderived either from lymphocytes or testicular tissue. Thefrequency of chromosomal aberrations in the 72 infertilepatients tested (62 azoospermic, 10 oligozoospermic) was16.6%, with a high percentage of gonosome anomalies.Additional andrological parameters (hormone values,cryptorchidism) failed to identify men at risk for havingmicrodeletions before the test. Our ﬁndings support therecommendation to performgenetic defect screening amonginfertile men before their enrolment in an intracytoplasmicinjection/in-vitro fertilization programme.Key words: infertility/karyotype/microdeletions/testisIntroductionAmong couples with fertility problems, male factor is theaetiology in about 50% of cases (de Kretser, 1997). Inthese instances, oligozoospermia or azoospermia (AZO) arefrequently observed. Genetic macroscopic defects (detectedby cytogenetic methods) and genetic microscopic defects(detected by molecular biology methods) have been identiﬁedin such infertile patients (Jaffe and Oates, 1994). The geneticinformation for the control of spermatogenesis carried onthe Y chromosome was ﬁrst stated by Tiepolo and Zuffardi(1976). Cytogenetic analysis of infertile men revealed that

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Genetic evaluation of infertile men

Spermatogenesis may be focal in non-obstructive azoospermia. The present study was conducted to determine whether the performance of multiple, rather than a single testicular sample contributes to obtaining spermatozoa in amounts sufficient for fertilization and cryopreservation in non-obstructive, azoospermic patients. Furthermore, the aim was to clarify the significance of location for retrieval from the testis in such cases. Three biopsies were taken from identical locations in 55 testes of 29 men with non-obstructive azoospermia: (i) the rete testis region, ii) the midline, and (iii) the proximal region of the testis. When sperm cells were detected, they were used for intracytoplasmic sperm injection (ICSI), and the remainder were then cryopreserved in as many aliquots as possible (adjusted for ICSI procedure). Spermatozoa were found in 28 testes (50.9%) of 18 men (62.1%). In the testes from which spermatozoa were obtained, they were present in three, two or one locations in 15 (53.6%), five (17.9%) and eight (28.6%) cases respectively. The possibility of finding spermatozoa was not influenced by the location in the testis. Multiple testicular sperm extraction is recommended in cases of non-obstructive azoospermia, since it may enhance diagnostic accuracy of absolute testicular failure and increase the number of sperm cells retrieved.

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Multiple testicular sampling in non-obstructive azoospermia--is it necessary?

A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes.

distinct, the former being the most common type of lymphoma during the reproductive period of life, with a peak incidence patients with non-Hodgkin's lymphoma or with Hodgkin's may be irreversible, but even if reversed, repair may take as disease during the years 1987-1997 who were referred for long as 20 years (Marmor and Duyck, 1995). New treatment semen cryopreservation. Pretreatment semen analysis, and regimens have increased patient survival substantially, so that hormonal profile were recorded at diagnosis and at least more patients at the reproductive age are living and are 1 year after completion of the treatment, and compared. confronted with infertility. Medical records for disease type, disease stage and treat- Semen cryopreservation is an element of management of ment protocols were related to long-term sperm outcome. lymphoma patients. This is due to the growing awareness of Hormonal concentrations were not predictive of post- physicians and patients themselves regarding the potentially treatment sperm concentration. In patients with localized devastating effect of the disease and its treatment on spermato- disease, initial sperm concentration and motility tended to genesis and future fertility. Patients who had a lymphoma were be preserved, compared with patients with widespread referred to our institute for semen cryopreservation before disease (P 0.016). In Hodgkin's disease patients, treat- commencing treatment. Previously, we studied the effect of ment with the adriamycin, bleomycin, vinblastine and lymphomas on spermatogenesis and assessed pretreatment dacarbazine (ABVD) protocol was superior to the mechlor- sperm quality (Botchan et al., 1997). The present study etamine, vincristine, procarbazine and prednisone with was conducted to determine the factors, either the disease ABV protocol regarding germinal toxicity (P 0.0008). characteristics themselves, or different treatment protocols, The post-treatment sperm outcome was better in patients directly related to semen concentration and motility deteriora- treated with local irradiation than in those who did not tion. In this study we assessed pre- and post-treatment sperm undergo irradiation (P 0.0027). No predictive tools for quality, reflected by sperm motility and concentration. Patients post-treatment fertility were found and, therefore, every were categorized by their disease (HD or NHL), disease stage patient with a lymphoma should have his semen cryopre- and treatment. Staging included distinction between patients served at diagnosis. with and without systemic symptoms: fever, weight loss and

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Follow-up of sperm concentration and motility in patients with lymphoma

Men diagnosed as having azoospermia occasionally have a few mature sperm cells in other ejaculates. Other men may have constant, yet very low quality and quantity of sperm cells in their ejaculates, resulting in poor intracytoplasmic sperm injection (ICSI) outcome. It has not been conclusively established which source of sperm cells is preferable for ICSI when both ejaculate and testicular (fresh or frozen) sperm cells are available. It is also unclear whether there is any advantage of fresh over frozen sperm if testicular sperm is to be used. We used ejaculate, testicular (fresh or frozen) sperm cells, or both for ICSI in 13 couples. Five of these couples initially underwent ICSI by testicular sperm extraction, because the males had total azoospermia, and in later cycles with ejaculate sperm cells. Ejaculate sperm cells were initially used for ICSI in the other 8 patients, and later with testicular sperm cells. The fertilization rate was significantly higher when fresh or frozen-thawed testicular sperm cells were used than when ejaculated sperm cells were used. Likewise, the quality of the embryos from testicular (fresh and frozen) sperm was higher than from ejaculated sperm (65.3% vs 53.2%, respectively, P < .05). The use of fresh testicular sperm yielded better implantation rates than both frozen testicular sperm and ejaculate. Therefore, fresh testicular sperm should be considered first for ICSI in patients with virtual azoospermia or cryptozoospermia because of their superior fertility.

Ron Hauser

Papers

Genetic evaluation of infertile men

Multiple testicular sampling in non-obstructive azoospermia--is it necessary?

A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes.

Follow-up of sperm concentration and motility in patients with lymphoma

Virtual azoospermia and cryptozoospermia--fresh/frozen testicular or ejaculate sperm for better IVF outcome?