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- Review -

Gonadal damage and options for fertility preservation in female and male cancer survivors

Theodoros Maltaris¹, Heinz Koelbl¹, Rudolf Seufert¹, Franklin　Kiesewetter², Matthias W. Beckmann³, Andreas Mueller³,Ralf Dittrich³

¹Department of Obstetrics and Gynecology, Johannes Gutenberg University, Mainz D-55124, Germany

²Division of Andrology, ³Department of Obstetrics and Gynecology, Erlangen University Hospital, University of Erlangen-Nuremberg, Erlangen, Germany

Abstract

It is estimated that in 2010, 1 in every 250 adults will be a childhood cancer survivor. Today, oncological surgery, radiotherapy and chemotherapy achieve relatively high rates of remission and long-term survival, yet are often detrimental to fertility. Quality of life is increasingly important to long-term survivors of cancer, and one of the major quality-of-life issues is the ability to produce and raise normal children. Developments in the near future in the emerging field of fertility preservation in cancer survivors promise to be very exciting. This article reviews the published literature, discusses the effects of cancer treatment on fertility and presents the options available today thanks to advances in assisted-reproduction technology for maintaining fertility in male and female patients undergoing this type of treatment. The various diagnostic methods of assessing the fertility potential and the efficacy of in vitro fertilization (IVF) after cancer treatment are also presented. (Asian J Androl 2006 Sep; 8: 515_533 )

Keywords: reproduction; cryopreservation; male infertility; semen preservation; fertility preservation; cancer treatment; ovarian tissue

Correspondence to: Dr Theodoros Maltaris, Department of Obste-trics and Gynecology, Mainz University Hospital, Langenbeckstrasse 1, D-55124 Mainz, Germany.

Tel: +49-6131-177-995, Fax: +49-6131-174-321
E-mail: maltaris@uni-mainz.de
Received 2006-03-10 Accepted 2006-06-05
DOI: 10.1111/j.1745-7262.2006.00206.x

1  Introduction

Today, approximately 1 in 700 young adults is a cancer survivor [1], whereas it is estimated that in 2010, 1 in every 250 adults will be a childhood cancer survivor [2]. In the USA alone, more than 20 000 children and young people of reproductive age are exposed to known mutagens in the form of chemotherapy and/or radiotherapy for cancer every year [3]. Women today are using better methods of contraception and are delaying childbearing for social or financial reasons, so that increasing numbers of women are anxious to preserve their fertility when early-stage cancers are discovered [4_6]. Also, patients with nonmalignant autoimmune diseases, such as rheumatoid arthritis or systemic lupus erythematosus and hematological diseases are being treated successfully with chemotherapy or radiotherapy [7].

Surgery, radiotherapy and chemotherapy might achieve relatively high rates of remission and long-term survival, but are often detrimental to fertility. Quality of life is increasingly important to long-term survivors of cancer, and one of the major quality-of-life issues is the ability to produce and raise normal children.

This article reviews the published literature, discusses the effects of cancer treatment on fertility, and presents the options available for maintaining fertility in male and female patients undergoing such treatment. The various diagnostic methods of assessing the fertility potential and the efficacy of in vitro fertilization (IVF) after cancer treatment are also presented.

2  Ovarian anatomy and physiology

The peak number of oocytes in the ovary, approximately 6.8 × 10⁶, occurs at 5 months’ gestation. After this point, there is no further proliferation of germ cells, and progressive atresia occurs. At birth, the number of oocytes decreases to 1_2 × 10⁶, and at puberty there are only 300 000 left. Approximately 300_500 of these follicles will develop into mature oocytes, whereas the rest will become atretic. In women aged 51 years, the average age of natural menopause in the developed countries, there are approximately 1 000 left [8].

In healthy women, accelerated atresia of the oocytes begins at approximately 37.5 years of age, associated with an increase in the level of follicle-stimulating hormone (FSH) [9]. As atresia continues, both the number and quality of oocytes fall below a critical level, and the rate of aneuploidy increases. This process leads to a greater risk of spontaneous abortion once pregnancy occurs. This central principle of age-dependent follicle depletion has been challenged by recent data suggesting that ovarian stem cells are present in mice and could presumably lead to the replenishment of follicles [10]: a theory that cannot be supported in humans, at least.

Premature ovarian failure, defined as menopause before the age of 40 years or hypergonadotropic amenorrhea, occurs in up to 0.9% of women in the general population.

There are three major developmental phases in the life cycle of the ovary [11]:

1 The phase of embryogenesis: In this phase, populations of primordial germ cells and somatic cells become an integrated ovary mass containing oocytes and granulosa cells located within primordial follicles. The first phase of oocyte maturation starts in utero, and is gonadotropin-independent.

2 The pubertal and adult phase: The gonadotropin-dependent folliculogenesis begins, in which oogenesis, granulogenesis and thecogenesis occur as a recruited primordial follicle grows and develops into the preovulatory follicle, or dies by atresia.

3 The postmenopausal phase: Despite the high circulation of gonadotropins the ovarian cortex is thin and usually devoid of follicles.

There are five phases of follicular development in the adult ovary [11]:

1 The preantral phase: The earliest phase of follicular growth is characterized by an increase in follicular diameter of the primary oocyte and the formation of a granulosa cells layer. The initiation of this phase is hormone independent.

2 The antral phase: The preantral follicles either die by the process of atresia or develop, when there are adequate levels of FSH and luteinizing hormone (LH) to the antral follicles. These are distinguished by a further increase in follicular size and the development of the follicular antrum. There is now a significant increase in androgen and estrogen production.

3 The preovulatory phase: This is the shortest and most dramatic phase of follicular growth. The antral follicles will die unless a brief surge of gonadotropin coincides with the appearance of LH receptors on the outer granulosa cells. This surge of LH causes terminal growth in both the follicle cells and the oocyte and the chromosomes progress from the arrested first meiotic prophase to the second metaphase.

4 The ovulation: The oocyte, triggered by the LH, transforms into a mature egg, which is secreted into the oviduct to await fertilization.

5 The corpus luteum: The follicle luteinizes into the corpus luteum, which dies by a process termed luteolysis if implantation does not occur.

3  Testicular anatomy and physiology

The testis consists of the seminiferous (or germinal) epithelium, arranged in tubules, and endocrine components (testosterone-producing Leydig cells) in the interstitial region between the tubules. The seminiferous tubules contain the germ cells, which consist of stem and differentiating spermatogonia, spermatocytes, spermatids and sperm, and the Sertoli cells, which support and regulate germ cell differentiation.

In men, germinal stem cells exist in the testicles from the time of birth, but do not develop into the haploid gametes (spermarche) until the boy reaches puberty. In the prepubertal testis, there is a steady turnover of early germ cells that undergo spontaneous degeneration before the haploid stage is reached, which is probably the reason why the prepubertal testis is very vulnerable to cytotoxic therapy. In the mature testicle, the germinal stem cells undergo continual self-renewal and differentiation into mature spermatozoa within approximately 67 days throughout life. Therefore, there are always germ cells in various developmental stages in the testicles [12].

The loss of germ cells has secondary effects on the hypothalamic-pituitary-gonadal axis. Inhibin secretion by the Sertoli cells declines, and, consequently, serum FSH levels rise. Testicular blood flow is reduced, resulting in less testosterone being distributed in the circulation. Therefore, levels of LH increase to maintain constant serum testosterone levels [12].

The eventual recovery of sperm production after cancer treatment depends on the survival of the spermatogonial stem cells and their ability to differentiate. If treatment is limited to cytotoxic agents that do not kill stem spermatogonia, normospermia is usually restored within 3 months after cytotoxic therapy [13].

4   The effect of cancer treatment on female ferti-lity

4.1 Radiotherapy-induced ovarian damage

The degree and persistence of radiation induced gonadal damage depends on the cumulative dose, the irradiation field and the patient’s age, with older women being at greater risk of damage [14].

Exposure of the ovaries to high radiation doses, in the range of 30_700 Gy, as is the case for treatment of cervical and rectal cancer, and with craniospinal radiotherapy for central nervous malignancies can cause a mutagenic, embryotoxic, embryolethal and teratogenic effect [3]. This can also happen when the pelvic lymph nodes are irradiated for hematological malignancies, such as Hodgkin’s disease, and with total body irradiation before bone marrow transplantation, so it is recommended that, when possible, the gonads should be shielded, the radiation field restricted, or when indicated the ovaries should be surgically relocated away from the radiation field (oophoropexy) [3].

Depletion of primordial follicles in mouse ovaries in a dose-related fashion using increasing radiation doses of 0.1, 0.2 and 0.3 Gy is demonstrated by Gosden et al. [15]. Exposure to high doses of radiotherapy caused sterilization, whereas lower doses that cause only partial depletion of the primordial follicle reserve resulted in premature ovarian failure [15].

The radiation dosage necessary for loss of ovarian function has been examined in many studies. Thibaud et al. [16] show that total body irradiation (TBI) of < 10 Gy administered in a single dose before puberty causes a high ovarian failure rate (55_80%), whereas fractionated TBI is less toxic to the ovaries even at higher doses. With fractionated TBI of > 15 Gy, ovarian fai-lure is present in all cases. Socie et al. [17] note that that the incidence of pregnancy after TBI was less than 3% and that the outcome for recovery was more favorable if the patient was prepubertal and the radiation was delivered in several fractions.

Wallace et al. [18] estimate the dosage at which half of the follicles are lost in humans (LD₅₀) to be 4 Gy. Lashbaugh and Casarett [19] observe that women under 40 years of age are less sensitive to radiation-induced ovarian damage, with an estimated dose of 20 Gy being required to produce permanent ovarian failure in comparison with 6 Gy in older women.

Chiarelli et al. [20] observe a dose-dependent and distribution-dependent relationship between the risk of premature ovarian failure and the total dosage of abdominal pelvic irradiation: with doses < 20 Gy, the relative risk was 1.02; with irradiation of 20_35 Gy, the relative risk increased to 1.37; and with doses > 35 Gy, the relative risk of premature ovarian failure was 3.27. The percentage of women who suffered from infertility correlated with increasing dosages of abdominal pelvic irradiation: treatment doses of 20_35 Gy caused a 22% rate of infertility, and doses > 35 Gy caused a 32% rate of infertility.

Uterine irradiation is associated with infertility, spontaneous pregnancy loss and intrauterine growth retardation ã 2006, Asian Journal of Andrology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All rights reserved.

[21]. Direct effects on the uterus after irradiation include irreversible changes in the uterine musculature and blood flow, as well as hormonal-resistant endometrial insufficiency [22, 23]. A study by Holm et al. [24] shows that young women exposed to TBI also suffer impaired uterine growth and later require sex steroid replacement therapy. A review by Critchley and Wallace [25] indicates that physiological sex steroid replacement therapy might improve uterine characteristics in some patients after irradiation at a young age.

There are also increased rates of obstetric complications in patients who have undergone radiotherapy in comparison with the general population, including spontaneous abortions (38% vs. 12%), preterm labor (62% vs. 9%) and low-birthweight infants (62% vs. 6%). However, as long as radiation is not administered during pregnancy, there is no risk of subsequent teratogenicity [26, 27]. These findings confirm research on women exposed to the atomic bomb and on offspring conceived and born to them after exposure, which show that the incidence of spontaneous abortion is greater, but that the children do not suffer a higher rate of mutations or major congenital anomalies in comparison with the normal population [28].

Swerdlow et al. [29] confirm that there was no excess of stillbirths, low birth weight, congenital malformations, abnormal karyotypes or cancer in the offspring of women treated for Hodgkin’s disease. However, Fenig et al. [30] report an increase in low birth weight and spontaneous abortions, especially if conception occurred less than a year after radiation exposure. They advise delaying pregnancy for a year after the completion of radiotherapy.

Radiation of the hypothalamic_pituitary area for brain tumors in excess of 30 Gy often causes early or even precocious puberty [31]. In addition, children who have received larger doses of cranial irradiation are at risk of developing hypogonadotropic hypogonadism in time [32].

4.2 Bone marrow transplantation

Bone marrow transplantation (BMT) has come into widespread use in last 30 years in the treatment of oncohematological malignancies. The conditioning regimens used for BMT include high-dose chemotherapy, with or without body irradiation. In a survey of 38 000 male and female patients who had received high-dose chemotherapy or TBI with allogeneic/autologous stem cell transplantation, the fecundity rate was found to be extremely low, with only 129 pregnancies reported [33]. Many studies have confirmed the extremely high risk of persistent ovarian failure in women who undergo BMT [34]. Growth and sexual development are impaired in children, and sterility is common in adults [14], so every effort to preserve fertility should be undertaken before BMT.

4.3 Chemotherapy-induced ovarian damage

Chemotherapeutic drugs act by interrupting vital cell processes and arresting the normal cellular proliferation cycle. The risk of chemotherapy-related amenorrhea is related to the patient’s age, the specific chemotherapeutic agents used, and the cumulative dosage administered [35].

4.3.1 Age

Women over 38 years of age have a higher incidence of complete ovarian failure and permanent infertility in comparison with younger women, who have a larger primordial follicle reserve [14, 36].

4.3.2 Cytotoxic drugs

Alkylating agents These substances have a severe effect on human fertility. Known effects are ovarian fibrosis and follicular and oocyte depletion [37]. According to Meirow [38], alkylating agents, such as cyclophosphamide, involve the greatest risk of inducing ovarian failure among all the chemotherapeutic agents (odds ratio 3.98) in comparison with unexposed patients.

Cisplatin and analogs Meirow [38] estimates that cisplatin causes ovarian failure, with an odds ratio of 1.77. Studies of cisplatin treatment in female mice demonstrate the induction of different types of chromosomal damage [39].

Vinca alkaloids These substances are known aneuploidy inducers. According to Meirow, the odds ratio for ovarian failure is approximately 1.0 [38]. Many animal experiments have shown high levels of aneuploidy in oocytes exposed to vinblastine [40], which means that these damaged oocytes could produce malformed fetuses.

Antimetabolites Insufficient data are available on the effects of antimetabolites on female germ cells.

Anthracycline antibiotics Adriamycin and bleomycin are female-specific mutagens and have been shown to induce dominant lethal mutations in maturing/preovulatory oocytes in female mice [39].

Meirow et al. [41] demonstrate in animal experiments that regular menses and normal reproductive outcome after chemotherapy are not certain indicators of whether the ovarian follicular reserve has survived the treatment unaffected. They recommended that patients who recover from ovarian failure after high-dose chemotherapy or radiotherapy treatments should not delay childbearing for too many years. These patients should try to conceive after a disease-free interval of few years, but not < 6_12 months after treatment, because of the possible toxicity of the treatment for growing oocytes [38, 42].

Regarding the teratogenic effects of chemotherapy, studies that have monitored pregnancies in women exposed to chemotherapy before conception have not re-gistered increased rates of miscarriage or congenital abnormalities in comparison with the general population. Because these pregnancies occurred long after treatment had ceased, it can be assumed that there are correction mechanisms within the oocyte or that there are undetected miscarriages as a result of dominant lethal mutations at a very early stage [42].

5  The effect of cancer and cancer treatment on male fertility

Infertility is a major concern for young men of reproductive age undergoing chemotherapy, radiotherapy or surgery, as most of these regimens can cause sterility. Several studies have indicated that some cancer patients have a reduced fertility potential even before starting treatment [43, 44]. This infertility can result from anatomical changes (e.g. hypogastric plexus damage leading to retrograde ejaculation), primary or secondary hormonal imbalance, or damage to germinal stem cells or supporting cells. These changes can result in compromised sperm numbers, motility, morphology or DNA integrity [4].

Malignancy is also associated with an increased catabolic state, malnutrition, an increase in stress hormones, and a decrease in pituitary gonadotropin levels, which can also have an impact on fertility [45].

5.1 Testicular cancer

Testicular cancer is the most common malignancy in young men, with a well-known association with abnormalities of spermatogenesis [43]. A statistically significantly lower sperm count was found in 83 patients with testicular germ cell cancer than in healthy men [46]. The exact mechanism responsible for this decreased sperm quality is unknown. One possible explanation might be a preexisting defect in germ cells, leading to both cancer and defective spermatogenesis, after bilateral undescended testis, genetic abnormalities or exposure to abnormal hormonal levels in utero, for example. Another explanation might be a local effect of the tumor itself, caused by the paracrine action of the tumor’s secretory substances.

Cancer might also alter the process of spermatoge-nesis through a hormonal imbalance. The general systemic effects might lead to oversecretion or underse-cretion of hormones by the endocrine glands, or the tumor might secrete its own hormones affecting spermatogenesis hormones, such as b-human chorionic gonadotropin and a-fetoprotein [47].

5.2 Hodgkin’s lymphoma and leukemia

Hodgkin’s lymphoma has also been associated with pretreatment impairment of spermatogenesis [48]. A study in 158 patients with Hodgkin’s disease reports that elevated erythrocyte sedimentation rates and advanced disease stages are prognostic factors for severe fertility damage [49]. In another study, 70% of patients with Hodgkin’s lymphoma had reduced levels of fertility before therapy, regardless of the disease stage or systemic symptoms [50].

Immunological processes that alter the balance between subpopulations of lymphocytes are also associated with spermatogenetic disorders induced by Hodgkin’s disease [51].

Hallak et al. [52] found that the pretreatment semen quality (the median motile sperm count and motility) was poor in patients with acute and chronic leukemia.

5.3 Radiation effects

Ionizing radiation has adverse effects on gonadal function in men of all ages. The degree and persistence of the damage is dependent on the dose, the treatment field and the fractionation schedule [53]. Sperm production is susceptible to damage at very low doses of irradiation (> 1.2 Gy), but as Leydig cells are more resistant to damage from the radiotherapy than the germinal epithelium (function is usually preserved up to 20 Gy in prepubertal boys and 30 Gy in sexually mature men), progression through puberty with normal testosterone levels is common, despite a severe impairment of spermatogenesis [54]. Doses of more than 4 Gy can cause permanent damage to spermatogenesis [55]. Sperm counts are typically at their lowest 4_6 months after treatment is completed, and a return to pretreatment levels usually occurs in 10_24 months, with longer periods being required for recovery after higher doses [56]. TBI as a conditioning regimen for stem cell transplantation causes permanent gonadal failure in approximately 80% of men [17].

Recovery of spermatogenesis takes place from surviving stem cells (type A spermatogonia) and is dependent on the dose of radiation. Complete recovery, as indicated by a return to pre-irradiation sperm concentrations and germinal cell numbers, takes place within 9_18 months following radiation with 1 Gy or less, 30 months for 2_3 Gy, and 5 years or more for doses of 4 Gy and above [57].

In many cases, men who regain spermatogenesis after cancer treatment have low sperm counts and motility and an increased rate of chromosomal abnormalities [58]. These effects appear to be dose-dependent, with an apparent threshold [59], and persist for up to 3 years after radiotherapy, so that contraception for a period of 1_3 years is recommended after testicular irradiation.

5.4 Chemotherapy effects

Cytotoxic chemotherapy can cause gonadal injury, and the nature and extent of the damage depends on the drug administered, the dosage received, and the age of the patient [3]. In general, cytotoxic treatment targets rapidly dividing cells, and it is therefore not surprising that spermatogenesis can be impaired after treatment for cancer. The exact mechanism of the damage is uncertain, but it appears to involve depletion of the proliferating germ cell pool, by killing cells not only at the stage of differentiating spermatogonia but also stem cells themselves. In addition, stem spermatogonia that do survive fail to differentiate further [60]. Chemotherapy appears to lower healthy sperm counts in cancer survivors, but after an adequate period of therapy, small studies suggest that the DNA integrity of sperm is reestablished at a level similar to that in age-matched control individuals [61, 62]. Several studies report that most offspring of cancer survivors do not have any adverse effects resulting from preconception exposure to therapy [63, 64].

5.4.1 Alkylating agents

These cause depletion of the germinal epithelium in the testes and aplasia of germinal cells, resulting in severe oligospermia or azoospermia within 90_120 days of treatment [65], with poor long-term recovery [43]. In another study, most men had not regained spermatogenesis 4 years after cyclophosphamide treatment; those men who did regain spermatogenesis did so after an average interval of 31 months [66]. Alkylating agents are mutagenic in all stages of maturation of male human germ cells, but these agents do not cause transmissible chromosomal translocation or aneuploidy in stem cells [67]. The vast majority of men receiving procarbazine-containing regimens for the treatment of lymphomas are rendered permanently infertile [57].

5.4.2 Cisplatin and analogs

In studies in male mice, cisplatin induced chromosomal aberrations in spermatocytes, as well as differentiating spermatogonia immediately after the treatment. However, long after exposure, the transmission of such effects was found to have decreased substantially by the time the exposed spermatogonia matured [68].

5.4.3 Vinca alkaloids

In men, these agents arrest spermatogenesis and might also affect the motility of mature spermatozoa [3]. Vinblastine is cytotoxic to primary spermatocytes, while spermatogonia and preleptotene spermatocytes are relatively resistant [69].

5.4.4 Antimetabolites

These agents act on rapidly dividing cells (i.e. in the later stages of spermatogenesis) and induce dominant lethal mutations. 5-fluorouracil and 6-mercaptopurine cause chromosomal aberrations [70]. In Meirow [71], long-term administration of 6-mercaptopurine in low doses to male mice induced a high embryonic resorption rate in pregnant female mice mated with the exposed male mice.

5.4.5 Topoisomerase interactive agents

These agents are cytotoxic to all spermatogonial stages. The mutagenic effects of Adriamycin (doxorubicin) have been demonstrated in mice spermatocytes [72]. Administration of bleomycin to male mice also induces chromosomal anomalies in spermatogonia and spermatocytes [73].

5.4.6 Combination chemotherapy

The MOPP regimen (mechlorethamine, oncovin/vincristine, procarbazine and prednisone), used for Hodgkin’s disease, can cause azoospermia in 90% of men up to 4 years after therapy, as well as an increased frequency of aneuploidy for up to 18 years after treatment [74]. Sperm chromosomal anomalies were also assessed in testicular cancer patients before, during and after BEP (bleomycin, etoposide and cisplatin) chemotherapy [75]. Sperm aneuploidy was evaluated (using fluorescence in situ hybridization) in male patients with Hodgkin’s disease who were treated with NOVP (novantrone/mitoxantrone, oncovin/vincristine, vinblastine and prednisone) chemotherapy [76]. Current treatment of Hodgkin’s lymphoma in children includes chemotherapy with ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine), which appears to be less gonado-toxic, and ChlVPP (chlorambucil, vinblastine, procarbazine and prednisone), a treatment that is known to cause gonadal damage, especially in men [21].

5.5 Effects of oncological surgery

Surgical intervention for cancer therapy, such as bladder neck or prostate resection, bilateral retroperitoneal lymphadenectomy, or extensive pelvic surgery, might result in anejaculation as a result of retrograde flow of semen into the urinary bladder. Modified nerve-sparing surgical improvements have reduced this adverse outcome without compromising the efficacy of the procedure. Depending on the location of lymph nodes with metastases, retroperitoneal lymph-node dissection might involve a modified template of dissection (unilateral dissection below the inferior mesenteric artery, avoiding the lumbar sympathetic fibers and hypogastric plexus), preserving ejaculation in 50_85% of men. Similarly, improved surgical techniques in the treatment of bladder and prostate cancer avoid damaging the nerve fibers in the neurovascular bundles that innervate the penile corpora cavernosa. Consequently, 70_80% of men with radical prostatectomy or radical cystoprostatectomy maintain sexual function [77].

6  Fertility preservation options for female cancer patients

6.1 Ovarian transposition (oophoropexy)

Transposition of the ovaries out of the field of irradiation was described initially in 1958 [78]. The most common indications for this are Hodgkin’s disease, cervical and vaginal cancer, and pelvic sarcomas. The ovarian dose after transposition is reduced to approximately 5_10% of that in the in situ ovaries [79]. Lateral ovarian transposition is typically carried out by laparotomy, with division of the utero-ovarian ligament and tubes and with the ovaries being removed to the paracolic gutters so that they lie 3 cm above the upper border of the field: a safety margin that maintains ovarian function [80]. Ovarian failure might result if the ovaries are not removed far enough, or if they migrate back to their original position.

Ovarian transposition is currently also being carried out laparoscopically [81], which offers the following advantages: there are fewer adhesions, radiotherapy can be initiated immediately postoperatively, and the laparoscopy can be repeated if postoperative assessment of the ovaries shows that the radiation dose is still likely to be significant. Various laparoscopic surgical procedures have been described [82].

Various degrees of preservation of ovarian function and the ability to conceive after radiation treatment and oophoropexy have been reported, ranging from 16 to 90% [81, 82]. The variations are a result of the inability to calculate and prevent scatter radiation, concomitant use of chemotherapy, and the different radiation dosages used [7].

6.2  GnRH analogs

Blumenfeld and other researchers, although in small studies, demonstrate that GnRH agonists are well tolerated and might protect long-term ovarian function [83, 84]. Blumenfeld reports on probably the largest group of women (55 lymphoma patients) who were started on GnRH analogs 7_10 days before chemotherapy treatment. The rate of premature ovarian failure was 5% in the GnRH analog/chemotherapy group vs. 55% in the group receiving chemotherapy alone [83].

However, contradictory results have been published on the effects of GnRH agonists, and there has been intensive debate on the existence of FSH receptors in primordial follicles and GnRH agonist receptors in the human ovary [85]. Meirow [86] did not observe a protective effect of GnRH after ablative chemotherapy and radiotherapy in patients undergoing bone marrow transplantation. Waxman et al. [87] found that buserelin was not effective for fertility preservation in humans. However, it is possible that complete pituitary ovarian suppression was not achieved, which might be a necessary condition for these drugs to work.

The role of GnRH agonists in the treatment of gynecological cancers has been the subject of intense investigation. Approximately 50% of breast cancers, 70% of ovarian cancers, and 80% of endometrial cancers express GnRH and its receptor [88]. Native GnRH and GnRH agonists inhibit the proliferation of human breast cancer, ovarian cancer, and endometrial cancer cell lines in a dose-dependent and time-dependent manner [89]. Emons et al. [88] suggest that GnRH and its receptor are part of a negative autocrine system that might be used therapeutically to inhibit cell proliferation by the administration of GnRH analogs.

GnRH agonists are used today both in adjuvant treatment and in metastatic breast cancer for reversible medical castration to downregulate pituitary gonadotropin secretion, leading to suppression of ovarian estrogen production [90].

These data show that GnRH agonists can be safely used for fertility purposes. Prospective and randomized studies are currently being conducted to investigate the not yet proven efficacy of GnRH agonists, and the initial results of these are expected in the near future.

6.3  Sex steroids

The oral contraceptive pill has been investigated as an agent for suppressing the ovaries during chemotherapy and providing protection against cytotoxic agents. Chapman and Sutcliffe [91] report more follicles in ovarian biopsies from 3 patients who received combination oral contraceptive pills during chemotherapy than in those who did not. By contrast, Whitehead et al. [92] found no protective effect of combination oral contraceptive pills in patients who received chemotherapy for Hodgkin’s disease. One possible explanation for the varying results might be that the oral contraceptives do not manage to suppress the gonads completely.

6.4  Progesterone (P4)

In the rat, progesterone was found to have a protective effect when administered 1 week before the start of cyclophosphamide and during the treatment [93].

Familiari et al. [37] examined the protective effect of medroxyprogesterone acetate (MPA) on human primordial follicles exposed to cytotoxic drugs. Using electron microscopy, they showed that chemotherapy not only acutely damaged the ovary by reducing the number of follicles, but also chronically damaged the remaining follicular quality. MPA was unable to protect the ovary from early follicular atresia.

6.5  Apoptotic inhibitors

When mice oocytes were exposed to doxorubicin in vitro, they underwent a series of changes that produced apoptotic bodies [94]. Apoptosis also plays a significant role in the process of normal germ cell depletion [95], so that the existence of a genetic predetermined pathway has been suggested that can be aberrantly activated by chemotherapeutic drugs [96]. As a logical consequence, the use of apoptosis inhibitors could potentially stop the apoptotic process and protect the patient from premature ovarian failure.

The use of sphingosine-1-phosphate, a known apoptosis inhibitor, in mice treated with doxorubicin, was found to protect the oocytes from apoptosis. Also, the oocytes of mice that lacked the enzyme to generate ceramide and acid sphingomyelinase, early messengers in the apoptosis sequence, are more resistant to doxorubicin-induced apoptosis [97]. In Paris et al. [98] Sphingosine 1-phosphate preserved the fertility of irradiated female mice without any genomic damage for the offspring.

These studies show that these agents are promising but still at a very early experimental stage.

6.6  Cryopreservation of embryos

The most successful approach to fertility preservation is embryo cryopreservation, with delivery rates per embryo transfer using cryopreserved embryos reported to be in the range of 18_20% [99, 100]. However, this approach requires ovarian stimulation and consequent IVF and a participating male partner, although frozen sperm from a donor might also be used. Therefore, this option is not applicable to prepubertal adolescent girls.

In patients with estrogen-sensitive cancers (e.g. breast cancer) the use of the common stimulation agents for IVF purposes should be avoided. In such cases, antiestrogens, such as tamoxifen [101], or aromatase inhibitors [102] can be used, even if these regimens are less effective.

In a study by Oktay et al. [101] regarding the outcome of IVF in hormone sensitive breast cancer patients, tamoxifen (40_60 mg) was started on day 2 or 3 of the cycle and was administered daily for 5_12 weeks. The control group consisted of patients with an unstimulated IVF cycle. The tamoxifen group had a significantly higher number of mature oocytes, peak estradiol and embryos (mean of 1.6 embryos vs. 0.6 embryos) than the natural cycle group. They also reported the first pregnancy from cryopreserved embryos generated after tamoxifen stimulation.

Letrozole, an aromatase inhibitor, has been introduced as a promising ovulation induction agent [103]. Many groups are currently testing the feasibility of ovarian stimulation with aromatase inhibitors in breast and endometrial cancer patients. The patient is stimulated with gonadotropins, and an aromatase inhibitor is simultaneously introduced to reduce serum estradiol levels. Oocyte development is unaffected. A luteinizing hormone_releasing hormone antagonist is also used to prevent a premature luteinizing hormone surge [104]. Oktay et al. [105] compared the combination of tamoxifen or letrozole with FSH for stimulation in women with breast cancer, with very promising results.

Holzer et al. [106] report that aromatase inhibitors are as effective as, or superior to, clomiphene citrate for ovulation induction and in superovulation. However, their role in IVF remains to be determined.

6.7  Cryopreservation of oocytes

Oocyte banking is more problematic than cryopre-servation of sperms or embryos. The first obstacle is the sensitivity of oocytes to chilling, probably because of the sensitivity of the spindle apparatus and the higher lipid content of the cells. Cooling and exposure to cryoprotective agents (CPA) affect the cytoskeleton and might aggravate the already high incidence of aneuploidy in human oocytes [107]. Exposure to CPA also causes "hardening" of the zona pellucida, so that all oocyte cryopreservation protocols involve intracytoplasmic sperm injection (ICSI) as a precaution. Fertilization has to be carried out approximately 3_5 hours after thawing while the oocyte remains fertile. Further disadvantages of this method are that cancer patients might not have more than one opportunity for oocyte harvesting before undergoing potentially sterilizing treatment, because a cycle of controlled stimulation requires several weeks and there is normally a delay of a few months before treatment cycles. The success of the method also depends on the total number of eggs harvested (< 10 oocytes means very slight chances of pregnancy). To date, more than 4 300 oocytes have been cryopreserved and more than 80 children have been born, mostly with the conventional slow-cooling method. The overall live birth rate per cryopreserved oocyte is approximately 2%, which is much lower than that with IVF using fresh oocytes [108].

6.8  Cryopreservation of ovarian tissue

A promising method of preserving fertility is cryopreservation of ovarian tissue [109, 110]. The idea of cryopreserving ovarian tissue is based on the finding that the ovarian cortex harbors primordial follicles that are more resistant to cryoinjury than mature oocytes, because the oocytes they contain have a relatively inactive metabolism and lack a metaphase spindle, zona pellucida and cortical granules [111]. The clinical indications are almost identical to those for the oocytes, but there are fewer logistical restrictions and a greater fertility potential because of the far larger number of oocytes preserved.

Ovarian tissue cryopreservation might be the only acceptable method for any prepubertal or premenarchal female patients receiving chemotherapy or pelvic radiotherapy [4]. Follicular viability after cryopreservation and thawing is demonstrated in several studies [112]. Most of the follicles that survive cryopreservation are primordial [113]. There are three ways of getting these follicles to develop to maturity. The first is autografting: either orthotopic, which has yielded the first two pregnancies after cryopreservation of ovarian tissue [114, 115]; or heterotopic: for example, in the forearm s.c. [116]. The latter method requires the use of IVF to achieve a pregnancy.

The second option is in vitro follicular maturation and IVF: a method that has already yielded pregnancies in animal experiments [117]. This method is not applicable to the human species, owing to the long period necessary for the primordial follicle to reach the maturation stage [118].

The third method involves xenografting human ovarian tissue into immunodeficient animals (mice with severe combined immune deficiency) and stimulating it to full follicular maturation [119, 120].

The risks of ovarian tissue cryopreservation include reimplantation of the primary tumor and malignant transformation [121]. Shaw et al. [122] were the first to report the transmission of lymphoma from a donor to a graft recipient with fresh and cryopreserved mouse ovarian tissue samples. However, most of the malignant diseases encountered during the reproductive years in humans do not metastasize to the ovaries, with the following exceptions: blood-borne malignancies such as leukemias, neuroblastoma and Burkitt’s lymphoma [121]. Kim [123] examined the risk of cancer relapse by transplanting frozen-thawed ovarian tissue from lymphoma patients into immunodeficient mice. None of the ovarian grafts from non-Hodgkin’s or Hodgkin’s lymphoma patients resulted in recurrences, whereas cancer spread was found in one of the five animals transplanted with lymph nodes from Hodgkin’s disease. Therefore, a histological assessment for micrometastases should always be carried out on a small portion of the harvested tissue before cryopreservation. Another risk is the possibility of malignant transformation of the cryopreserved tissue after transplantation. In rats, heterotopic autotransplantation of cryopreserved ovarian tissue into the spleen resulted in the development of sex cord stromal tumors [124].

6.9  Construction of reconstituted human oocytes (artificial gametes)

The construction of artificial gametes might be made possible by transferring the nucleus of somatic cells into enucleated oocytes and inducing chromosomal halving (haploidization). The procedure involves nuclear transplantation of the cancer patient’s somatic cell into an enucleated ooplast obtained from a donor. The chromosomes contained within this nucleus can be induced to undergo meiosis, yielding a haploid cell: a functional oocyte. This reconstructed oocyte can then be fertilized by ICSI, creating a de novo individual and not a clone of the nuclear donor [125].

Although this method aims to ensure a normal genomic contribution from both parents, it has been observed that chromosomal aberrations are frequent, so that at this point this method has only experimental interest [126].

7  Fertility preservation options for male cancer patients

7.1 Sperm cryopreservation

Semen cryobanking before chemotherapy, radiotherapy or surgery affecting the reproductive system is a widely available and inexpensive option that yields good results and provides a reasonable chance of establishing a pregnancy after cancer therapy [127]. Traditionally, the banking of at least three semen samples, with an abstinence period of at least 48 hours between the samples, has been recommended. Completion of the process usually requires 5_8 days. Additional samples and longer abstinence periods (72_96 hours) to achieve higher total sperm counts might also be considered [128].

Various methods of semen collection are available, such as testicular sperm extraction [129], microsurgical epididymal sperm aspiration, penile vibratory stimulation, and electroejaculation even for younger adolescents [130]. Even a suboptimal sperm quality in not an insurmountable limitation, because ICSI allows successful fertilization even with a single sperm obtained from semen or by testicular sperm extraction [131, 132].

It is of interest that only a small percentage of patients (< 10%) who bank their spermatozoa before chemotherapy or radiotherapy return for assisted reproduction [133]. There are many possible reasons for this: recovery or waiting for possible resumption of spermatogenesis, a short period after the original illness, anxiety regarding potential risks for the children, and uncertainty about long-term health and suitability to be parents [134].

Statistics suggest that most men being treated for cancer do not participate in sperm banking and that most oncologists do not consistently discuss this option with their male patients. However, awareness of the option of sperm banking has increased in the past 4_5 years, coinciding with the advent of ICSI [45].

7.2 Hormonal therapy

Cytotoxic treatment acts principally on rapidly dividing cells. As the testis has high levels of cellular activity, it is, therefore, prone to this type of damage, and gonado-toxicity might result. In view of this, it has been postulated that inducing testicular quiescence could prevent gonadal toxicity by making germ cells less vulnerable to the cytotoxic effects. Spermatogenesis and fertility can be restored in rodents following treatment with radiation or some chemotherapeutic agents, by suppressing testosterone with GnRH agonists or antagonists either before or after the cytotoxic insult [13, 135].

However, there are species differences in the testicular response to radiation and GnRH antagonist therapy, and the rescue protocols that work in rodents have failed in clinical studies [136, 137]. Also in two recent studies conducted in non-human primates, hormone suppression by GnRH antagonists failed to enhance spermatogenic recovery after radiation, also indicating that there are important differences in this process between rats and primates [138, 139]. In addition, this approach might be ineffective in children, as the proliferation of germ cells in prepubertal primates might be gonadotropin-independent [140].

Therefore, hormonal manipulation based on the suppression of this axis is unlikely to be protective in such patients receiving gonadotoxic treatment [141].

7.3 Testicular tissue cryopreservation

Although the prepubertal testis does not produce mature spermatozoa, it does contain the diploid stem-germ cells from which haploid spermatozoa will ultimately be derived, so that testicular tissue could be harvested before chemotherapy and cryopreserved [141]. After the patient is cured, the tissue could be thawed and the stored germ cells could be reimplanted into the patient’s own testes, where they would give rise to complete and normal spermatogenesis in the seminiferous tubules: a procedure known as germ cell transplantation [142]. The extent of spermatogenesis depends on the number of transplanted stem cells and the quantity and quality of stem cell niches in the transplanted testis. Each donor-derived colony of spermatogenesis arises from the clonogenic proliferation and differentiation from a single spermatogonial stem cell (SSC). This is the only technology that has the potential to restore natural fertility from a patient’s own germ cells [143].

In a second procedure, testis tissue pieces could also be grafted to an ectopic site (e.g. under the skin) in cancer survivors or in immunodeficient animals (xenografting). Therefore, in contrast to the transplantation technique, when SSC are removed from their cognate niches and transplanted to the new niches in the recipient testes, testis grafting involves the transplantation of SSC with their niches intact. The grafted testicular tissue is revascularized in the ectopic site and produces complete spermatogenesis [143]. Successful stem cell transplantation has been reported in many species: mice [144], rats, monkeys and humans [145].

Alternatively, the stored cells could be matured in vitro until fertilization can be achieved by the use of ICSI. Although the technique of testicular germ cell harvesting, cryopreservation, and transplantation are effective in mice [146], there are considerable differences in human spermatogenesis.

The most important issue with autotransplantation is the risk of reintroducing malignant cells after retrans-plantation. The risk is greater with hematological cancers, as the testes can act as sanctuary sites for leukemic cells [147]. The technique of in vitro maturation of stem cells circumvents the risk of reintroducing malignant cells, making this procedure potentially highly beneficial in this patient group.

7.4 In vitro spermatogenesis

Maturing germ cells in vitro, stimulating their differentiation into spermatozoa, would be particularly useful in patients who have received profoundly gonadotoxic therapy in whom the supporting Sertoli cells are unable to support spermatogenesis. Although restoration of fertility after in vitro spermatogenesis has been reported [148], it involved maturation of the later stages of spermatogenesis rather than stem cells; in vitro maturation of diploid stem cells into haploid spermatozoa appears unlikely to become technically possible in the near future [149].

8  Testing for ovarian reserve

The term "ovarian reserve" denotes the available pool of primordial follicles in the ovary and is a major determinant of female fertility potential. There is certainly a need to assess the functional ovarian reserve in premenopausal patients with cancer. This information is important for the choice of the appropriate chemotherapy regimen and the correct strategy for fertility preservation before the cancer treatment, and can also serve as a guide for the prediction of premature menopause after cancer treatment. In general, ovarian reserve tests are either biochemical (basal or dynamic) or biophysical [150].

8.1 Biochemical (basal) ovarian reserve tests

8.1.1 Serum follicle-stimulating hormone

Basal serum FSH assessment is one of the longest-established parameters for estim, ating ovarian reserve. On day 3 of the menstrual cycle, serum FSH levels are usually less than 10 mIU/mL in most assays. FSH levels that are over 15 mIU/mL on day 3 suggest a decreased ovarian reserve and a reduced probability of pregnancy; if values exceed 20 mIU/mL, the probability of pregnancy is close to nil [8, 9]. Van Rooij et al. [151] observed an ongoing pregnancy in as many as 28% of women with regular cycles who had FSH levels of 15_20 IU/L, and there was a clear fall in the pregnancy rate regardless of age only when the FSH level was > 20 IU/L.

8.1.2 Estradiol

With the decline of the follicle pool, serum levels of E2 decrease [152]. However, the condensed follicular phase length in older women might be the result of an advanced follicular recruitment by cycle day 3. This early dominant follicle selection is expressed by high serum concentrations of estradiol. It has been shown in a population receiving assisted-reproduction treatment (in whom GnRH analogs were not administered) that increasing day 3 estradiol concentrations are associated with decreasing oocyte numbers and pregnancy rates [153]. Estradiol measurement is also useful when obtained concurrently with FSH levels, because values > 80 pg/mL indicate disrupted folliculogenesis, which does not allow accurate interpretation of FSH measurements [153]. Other authors have not found any correlation between estradiol concentrations and the ovarian reserve [154].

8.1.3 Inhibin B

Inhibins and activins are glycoproteins produced by the granulosa cells that belong to the transforming growth factor-b family. Inhibins have an inhibitory effect on pituitary FSH synthesis and secretion. Activins act as functional antagonists of inhibin to stimulate FSH synthesis and secretion [155]. Serum dimeric inhibin B is regarded as a direct measure of ovarian reserve, as it is mainly secreted by the granulosa cells of pre-antral follicles [156]. Low levels of both inhibin A and inhibin B are typical in women with premature ovarian failure and postmenopausal women [157]. Seifer et al. [158] suggest that a fall in the inhibin B concentration might be an earlier marker for limited ovarian reserve than an elevated FSH concentration.

8.1.4 Anti-Müllerian hormone

Another test of ovarian reserve is measurement of the level of anti-Müllerian hormone (AMH), which reflects the health of granulosa cells [159] and decreases with age in postmenopausal women [160]. AMH inhibits the recruitment of primordial follicles into the pool of growing follicles and also decreases the responsiveness of growing follicles to FSH [161]. There are findings that suggest that AMH is produced solely by antral follicles capable of growing and is independent of the gonadotropic status; serum levels of AMH might, therefore, represent both the quantity and quality of the ovarian follicle pool [162].

8.2 Ovarian stimulation tests (dynamic)

8.2.1 Clomiphene citrate challenge test

This test involves the administration of 100 mg clomiphene citrate on cycle days 5_9 and measurement of FSH concentrations on days 3 and 10 [163]. In women with a normal ovarian reserve, the increase in estradiol and inhibin production by the developing follicles should be able to overcome the estrogen antagonist effect of clomiphene on the hypothalamic-pituitary axis and suppress FSH levels back into the normal range by day 10. An exaggerated FSH response and/or an elevated basal FSH value are considered to be signs of diminished ovarian reserve. A recent comparison of the clomiphene citrate challenge test (CCCT) with basal markers (FSH, inhibin B, antral follicle count and estradiol) in 63 patients concluded that the increased burden placed on both patients and physicians by the CCCT is not justified [164].

8.2.2 Gonadotropin-releasing hormone agonist stimulation test

This test evaluates the estradiol serum concentration change from cycle day 2 to 3 after administration of a GnRH agonist, which causes a temporary increase in the pituitary secretion of FSH and LH, which, as a consequence, stimulates ovarian estradiol production [165]. However, the gonadotropin-releasing hormone agonist stimulation test has not been evaluated outside of groups receiving assisted-reproduction treatment and is relatively costly, and two early studies show only a limited ability of this test to differentiate between normal and diminished ovarian reserve [166, 167].

8.2.3 Human menopausal gonadotropin test

The most recent study using this test compared basal values of FSH, E2 and inhibin with hormonal and ultrasound parameters after 5 days’ stimulation with human menopausal gonadotropin [168]. However, the predictive value of the test is not high and it is rather expensive, so it has not become established in routine practice.

8.2.4 Exogenous follicle-stimulating hormone (FSH) ovarian reserve test

This test determines the increase in day 3 FSH and E2 serum concentrations over 24 h after administration of a standardized dose (300 IU) of purified FSH on day 3 [169]. A recent randomized and prospective study comparing basal and dynamic values found that the exogenous follicle-stimulating hormone ovarian reserve test was the best predictor for ovarian reserve [170]. 8.3 Biophysical ovarian reserve tests

8.3.1 Baseline assessment of the number of antral follicles by vaginal ultrasonography

The antral follicle count is defined as the ultrasound-detected number of antral follicles < 10 mm in diameter in the early follicular phase and is a reasonably good predictor of ovarian reserve [171]. An antral follicle count of less than five usually signifies a poorer prognosis [172].

8.3.2 Ultrasound measurement of ovarian volume

The ovary is a dynamic organ that changes in size during a woman’s life. From 0.7 cm³ at age 10 years, the ovarian volume increases to 5.8 cm³ at 17 years [173]; at the age of 40 years, the ovaries tend to decrease in size and they decrease even further after menopause [174]. Recently, a model has been proposed using the ovarian volume to predict reproductive age [175]. The main limitation of this method is the lack of data on age-dependent ovarian volume measurements in the general population.

8.3.3 Ovarian biopsy

There is contradictory evidence on the use of ovarian biopsy as an ovarian reserve test, as the follicular distribution is extremely heterogeneous [176]. Other authors draw attention to the use of the antral follicle count as the most predictive test of ovarian reserve [177].

9  Fertility potential in men after cancer treatment

In male patients, the assessment of gonadal function includes clinical assessment of pubertal progression, biochemical analysis of plasma gonadotropins and sex steroids, and, most important of all, a semen analysis. Testicular enlargement is the first sign of puberty in boys, followed by penis enlargement and the development of pubic hair. Many patients will have preserved Leydig cell function after gonadotoxic treatment and will, therefore, develop healthy secondary sexual characteristics. However, their testes might be of reduced size and consistency, with a loss of tubular space suggestive of diminished sperm production [178]. Men with mildly compromised Leydig cell function have been found to have normal plasma testosterone levels, but with slightly increased amounts of LH [139]. Inhibin B is secreted mainly from Sertoli cells in men and might be reduced after gonadotoxic chemotherapy, indicating reduced sperm production [179].

10  Efficacy of in vitro fertilization after chemotherapy

Little is known about the efficacy and safety of IVF in female patients who have been treated for cancer. In 2001, Ginsburg et al. [180] retrospectively examined 15 patients and found a poorer response to gonadotropins than in women with locally treated cancers, as well as a significantly diminished ovarian response to ovulation induction in comparison with patients who underwent IVF before cancer treatment. In a retrospective study, Dolmans et al. [181] examined the effect of chemotherapy directly before IVF treatment in a small number of patients (4 patients who underwent IVF in an interval between two chemotherapy regimens vs.7 patients who underwent IVF before starting chemotherapy) and report a dramatic reduction in the efficacy of IVF even after only one regimen. They recommend that in women whose cancer therapy can be delayed, IVF with embryo cryopreservation should be offered before chemotherapy rather than after it.

With regard to male cancer survivors, Sanger et al. [127] report live births after fertilization from cryopre-served semen. Agarwal et al. [182] report the outcome of assisted-reproduction technology in 29 male cancer survivors, in all cases with cryopreserved semen. A total of 87 cycles were carried out, with a mean pregnancy rate of 18.3% per cycle (7% after intrauterine insemination, 23% after IVF and 37% after ICSI) [182]. Schmidt et al. [183] examined the fertility outcome in 67 patients and found pregnancy rates of 14.8% after intrauterine insemination and 38.6% after ICSI.

11  Conclusion

Young cancer patients are still being poorly counseled with regard to the negative impact of the treatment on their fertility, as well as on their options for fertility preservation. This review focuses both on the effect of cancer treatments on fertility and on the various surgical and assisted-reproduction innovations that provide the patient with the possibility of future potential fertility. As the emerging discipline of fertility preservation is attracting steadily increasing interest, developments in the near future promise to be very exciting. However, in everyday routine work, better interdisciplinary cooperation between patients and pediatric oncologists, surgeons, immunologists and endocrinologists is necessary to provide individualized options for fertility preservation before surgical procedures or cancer treatment.

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