Abstract
The first live birth to occur after ovarian-tissue transplantation between two genetically different sisters was reported in 2011. Since this is an acceptable practice with monozygotic twins, there is no apparent reason to refrain from using it with genetically different sisters, especially if one of the sisters previously received bone marrow from the other, leading to complete chimerism (HLA compatibility) between donor and recipient, thus obviating the need for immunosuppressive treatment. This approach allows for natural conception, which could be important on moral, ethical or religious grounds.
Introduction
One of the consequences of cancer therapies, including radiation and chemotherapy, is gonadotoxicity. As effective treatments have rendered a number of malignancies curable, or have delivered long-term survival, posttreatment fertility has emerged as an important consideration for patients and their healthcare providers. Unfortunately, there are currently no definitive ways to limit the injurious effects of these treatments on gonadal function, other than shielding the gonads from direct exposure to ionizing radiation. Suppression of gonadotropin secretion may have a protective effect in some populations and with certain treatment regimens (e.g., alkylating agents), but the general efficacy of this intervention for preserving fertility remains uncertain [1].
Banking germ cells or embryos prior to treatment represent options for preservation of fertility. Sperm cryopreservation is a longstanding option for sexually mature males, and embryo cryo-preservation is an option for some women and couples. Egg freezing has become standard of care with improved cryopreservation techniques and recent successes with cryopreservation of ovarian cortex have been encouraging for women under 35. In general, options for gamete or embryo preservation are more complicated for women, and entail procedural risks and expense. Moreover, the technologies have not advanced to the point that female options for fertility preservation are as successful in outcomes as pretreatment sperm cryopreservation. The decision to pursue ovarian cortex or oocyte banking is complicated by the fact that the gonadal response to radiation and chemotherapy varies among the population, and there is, at present, no precise way to determine who will suffer irreversible damage and who will emerge from treatment with fertility intact, obviating the need for pretreatment interventions. Additionally, preserving the ovarian germ cell complement per se, while an important determinant of fertility, does not necessarily insure it. Furthermore, the inability to make predictions regarding the extent of posttreatment gonadal function impacts the design of research on interventions to spare fertility. Evaluations of such interventions could be conducted more efficiently with smaller sample sizes based on a more precise knowledge of subject risk for significant post-treatment gonadal dysfunction. Although the ability to generate germ cells from pluripotent stem cells has been demonstrated in laboratory animals, it has not been achieved with human stem cells[2].
In this chapter, we provide an updated framework for the prediction of gonadal function post-cancer therapy, noting progress in genetics, epigenetics, and population studies. We discuss issues that need to be addressed in future research with the aim of developing patient-specific algorithms that are predictive of post treatment fertility or infertility.
Predicting the weather requires knowing what the current weather conditions are in the specified location such as air temperature and humidity, what fronts and air masses are approaching and what other conditions might affect them. Local geography and previous weather patterns must also be taken into consideration. These are all elements that a meteorologist must assess before predicting future weather conditions. Using the weather prediction rubric, we can identify four general requirements for accurately predicting a future biological event such as preservation of ovarian function after cancer therapy:
1. The stability or flux of the current condition must be known (i.e., baseline fertility, germ cell complement, and rate of germ cell depletion).
2. Knowledge of what events or conditions could change the stability or rate of flux (i.e., types of treatment, dosing, duration).
3. The likelihood of those events or conditions happening (i.e., epidemiological data on post-therapy fertility by types of agents, dose and treatment duration, and age-related effects).
4. Potential for interactions of those events affecting outcome (i.e., host variables including differences in drug metabolism and action, intrinsic factors (i.e., genetic variation) affecting follicular complement, co-morbidities).
The Current State of Prediction of Future Ovarian Function Following Cancer Therapy
Alterations of the rate of follicle loss have been investigated in patients undergoing radiation and chemotherapy. However, other than age of the patient, the treatment and dosing of agents, specific predictors related to infertility have not been established. The current American Society of Clinical Oncology’s categorization of risk for gonadal dysfunction is broad and descriptive: (1) low, (2) medium, and (3) high. The medium-risk category encompasses risk of permanent cessation of menses (used as a surrogate marker of ovarian function) of between 30% and 80% [3]. The large range of probability in the medium-risk category is frustrating to physicians counseling patients; it also strongly suggests that there is substantial individual variation to ovarian susceptibility to damage by radiation and chemotherapy. Thus, while useful in understanding the effects of toxic agents on ovarian function, the existing classification of risk is not at all useful in predicting an individual’s future ovarian function. Recent population studies have only reinforced the lack of predictability of return of fertility for individuals after ovarian toxic therapies [4].
Genetic Risk Factors for Early Ovarian Senescence that Could Impact Ovarian Function after Cancer Treatment
In the last decade there has been a body of accumulated work that has identified genes that have a role in placing women at risk of earlier ovarian senescence. It is plausible that these risk alleles place women at an even greater risk of infertility after cancer treatment, although this notion has not been experimentally evaluated. Nonetheless, the wide range of ovarian compromise seen in women who have been treated for cancer, even with the same therapies, strongly suggests that the individual genetic variation controlling ovarian function may play a role in determining the level of ovarian damage.
Eggs reside in groups of cells called follicles within the ovary. Except for late in the reproductive life span, the majority of follicles are in a resting state at the primordial stage of development. The complement of primordial follicles has been called the resting pool [5] and represents ovarian reserve or the population of oocytes that have not yet become committed to the path of growth and ovulation. Since follicles (and eggs) are progressively lost from the resting pool over time by entering the growing pool or undergoing apoptosis, the ovarian reserve is constantly declining over time in women. In general, women with a diminished ovarian reserve are considered to have reduced fecundity [6].
Decay curves modeling the decline of the resting pool of follicles have been presented in the literature in mice and humans [7, 8]. In women, a bimodal decay was described, with a sharp increase in the rate of follicle loss at an average age of 38 years. However, a more recent model suggests that the rate of follicle loss or resting pool decay is one of slight constant acceleration throughout the perimenopause [9]. Investigators have explored the impact that radiation and chemotherapy have on shifting this “follicle population decay curve” to the left [10]. This model describes diminished ovarian reserve ensuing at an earlier age after the toxic treatments.
The ability to predict the consequences of decay in the germ cell population depends upon determining a woman’s current ovarian reserve, and there is presently no reliable method to accomplish this task. Antral follicle counts can vary in the hands of different observers, and secreted biomarkers such as anti-Mullerian hormone (AMH) and inhibin B suffer from lack of standardized assays and sufficient normative data. Recent data have demonstrated AMH to lack predictability of achieving pregnancy in general populations [11]. A key problem of secreted biomarkers is that while they can reflect the mass of growing follicle, they may not reflect the number of follicles remaining in the resting pool. Moreover, predicting how ovarian reserve might change over time is even more challenging.
There are several environmental and genetic conditions with clearly defined deleterious effects on ovarian reserve, including exposure to environmental/occupational toxins like 4-vinylcyclohexene diepoxide, heavy tobacco use, specific X-chromosome deletions and genetic variants (e.g., FMR1 premutations). When present, these might reasonably be expected to increase risk of ovarian failure following exposure to a gonadotoxic therapy. However, there are large number of genes involved in human ovarian development and follicular growth and their potential contributions to variability in response to cancer therapies are largely unknown. There are also other factors that could affect ovarian function when compounded with a gonadotoxic cancer therapy, whose impact is less well understood including exposure to bisphenols, dioxins, aniline dyes, immune issues, or even body mass (Figure 37.1).
Figure 37.1 Multiple interacting factors may contribute to ovarian reserve, including genetics, epigenetics, environmental factors (the exposome, which includes air pollutants, chemicals, medications, and ionizing radiation), behavioral factors (e.g., smoking), and nutrition and body mass index (BMI).The factors interact in complex and bidirectional ways. Variations in genes encoding enzymes that metabolize drugs or chemicals including components of cigarette smoke influence the ovarian exposure to gonadotoxic agents. These environmental factors can act via epigenetic mechanisms (e.g., DNA methylation) and the epigenetic marks can be transgenerational. Fat can influence ovarian reserve through the production of adipokines, the chronic inflammatory state associated with obesity, as well as serving as a depot for lipophilic agents with gonadotoxic activity. Genetics can influence BMI and behavior, as well as the expression of microRNAs, which are epigenetic regulators of gene expression
Control of Ovarian Development May Impact Future Fertility
Table 37.1 Selected genes and proteins with a reported role in follicle formation and growth in mammals
Germ cell differentiation
|
Germ cell migration and survival
|
Germ cell proliferation
|
Gonadal formation and colonization
|
Oocyte-specific early maturation
|
Follicle growth
|
Female fertility is dependent upon a series of critical ovarian developmental events that are controlled by a large number of genes on autosomes and the X chromosome. Variation in these critical genes which impact negatively on female fertility or fecundity might have even greater impact on women post-cancer treatment.
During human ovarian development, primordial germ cells must establish themselves in the epiblast of the post-gastrulation embryo and then migrate to the gonadal ridge. Once there, the presumptive oocytes must survive and become inhabitants of the cords of cells that will become granulosa cells. After the primordial follicle pool is established, it is at risk of diminution from both external and internal factors. Follicles can undergo destruction by toxins such as radiation or chemotherapy. Initiation of growth of larger groups (cohorts) of follicles can occur which would deplete the primordial pool more rapidly. However, to achieve fertility, follicles must grow in an orderly fashion in the proper endocrinologic milieu to allow ovulation of a mature fertilizable oocyte that can be fertilized and implant and grow within the uterus. Normal progression of folliculogenesis is just as important to continued ovarian function as maintaining ovarian reserve [12]. Anything that disrupts the process of gametogenesis from the initiation of embryonic germ cell formation to implantation of a fertilized embryo can reduce fertility.
The mouse genome has been investigated for candidate genes involved in ovarian fertility and 348 candidate genes involved in different stages of folliculogenesis have been identified [13]. No similar study has been performed in humans, but a number of genes have been clearly defined as being essential for human ovarian function and normal reproductive life span. A review of candidate genes associated with premature ovarian failure as well as linkage studies was recently presented by van Dooren et al. [14].
Human ovarian development requires the activity of autosomal genes and two functional X chromosomes. Although the requirement for two X chromosomes for ovarian development has long been recognized, the essential human X chromosome genes and their functions are largely unknown [15–20]. Cytogenetic studies have yielded clues as to the location of key ovarian function genes. Terminal deletions from Xp11 to Xp22.1 are associated with primary amenorrhea, and deletions from Xq13 to Xq27 are usually associated with primary amenorrhea or premature senescence. The region encompassing Xq13 to Xq26 is considered to be the “critical region” and this domain has been subdivided into two subregions Xq13–21 and Xq23–27.
The study of X chromosome gene variants and mutations has yielded a number of candidates for loci controlling germ cell complement. Among these is the fragile X syndrome gene (FMR1) on Xq27.3, which encodes an RNA-binding protein. Premutations in FMR1, represented by increased numbers of CGG trinucleotide repeats in the 5’-untranslated region, are well established to be associated with premature ovarian dysfunction.
The human orthologue of Drosophila diaphanous 2 (DIAPH2), a gene located on Xq22, encodes a protein involved in cytokinesis that, when mutated, causes sterility in flies. Premature ovarian failure associated with a translocation that disrupted the DIAPH2 gene has been reported. XPNPEP2, a gene located at Xp25, which encodes aminopeptidase P, was disrupted by a translocation associated with secondary amenorrhea. The zinc finger gene, ZFX, located on Xp22.1, is known to be important in murine ovarian development because heterozygous and homozygous mutations are associated with a reduced germ cell number. A mutation in the progesterone receptor membrane component-1 (PGRMC1) gene, located on Xq22-q24, has been found associated with premature ovarian failure. BMP15 located at Xp11.2 is a candidate gene for control of germ cell complement based on known ovine variants that result in follicular growth arrest in the Inverdale and Hanna sheep.
Autosomal genes also play important roles in controlling follicular dynamics. Mutations in the follicle stimulating hormone (FSH) receptor gene (FSHR), the ataxia telangiectasia gene (ATM), which is implicated in DNA repair and cell cycle control, the homeobox gene, NOBOX, NR5A1, also known as steroidogenic factor-1, a transcription factor in nuclear receptor family, and the forkhead transcription factor, FOXL2, are all associated with ovarian dysfunction or premature ovarian failure in certain populations. Mutations in FOXL2 cause blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), the type 1 form of which is associated with premature ovarian failure [21].
Mutations resulting in intranuclear aggregation and cytoplasmic mislocalization of FOXL2 are predictors of ovarian dysfunction. Polymorphisms in the gene encoding Inhibin alpha subunit (INHA) have been associated with premature ovarian dysfunction in some studies, but others dispute this association [22, 23].
Mutations in the catalytic subunit of the mitochondrial DNA polymerase gene, POLG, have been reported to segregate with premature ovarian dysfunction and ophthalmoplegia [24].
There has been one genome-wide linkage scan in a Dutch family that identified a region on chromosome 5 as a possible locus for familial premature ovarian dysfunction [25]. Comparative genomic hybridization profiling in a group of 99 Caucasian women with pre-mature ovarian failure [26] resulted in identification of 8 regions with copy number variations (1p21.1, 5p14.3, 5q13.2, 6p25.3, 14q32.33, 16p11.2, 17q12, and Xq28).
It is evident that larger genomic studies conducted in multiple populations are needed to identify gene variants associated with premature ovarian dysfunction and mutations across biogeographical ancestry.
Genes that have been associated with early ovarian senescence observed in isolated family groups or individuals are listed in Table 37.2. Although it may be assumed that women inheriting such genes or even groups of polymorphisms might have an earlier cessation of ovarian function, no markers have yet been shown to be predictive for individual fertility. There have been several promising recent studies correlating specific gene variants/mutations with markers of diminished ovarian reserve [26–36]. However, prospective studies validating these correlations are lacking at this time.
Table 37.2 Genes associated with early ovarian senescence in humans
| Autosomal | X-linked |
|---|---|
| • FOXL2 | • FMR1 |
| • NOBOX | • GDF-9 |
| • FSHR | • FMR2 |
| • AIRE | • DIAPH2 |
| • ATM | • XPNPEP2 |
| • POLG | • BMP-15 |
| • NR5A1 | |
| • MSH5 | |
| • NOG | |
| • INHA | |
| • FOXEI | |
| • B-glycan | |
| • PTHB1 | |
| • AR | |
| • DMC1 | |
| • ESR1 | |
| • PCMT1 | |
| • MCKDHB | |
| • ASCL6 | |
| • PGRMC1 | |
| • FIGLA | |
| • DMCI | |
| • SALL4 | |
| • PTEN | |
| • TGFMR3 | |
| • MeM8 | |
| • MCM9 | |
| • STAG3 | |
| • SYCE1 | |
| • NANOS3 | |
| • HMF1 |
Special Cases Resulting in Reduced Ovarian Reserve
Though data are accumulating regarding age at natural menopause, there is a strong rationale for genetic testing of individuals who are likely to have X chromosome structural abnormalities or genetic variants that diminish ovarian reserve. These include variants that are known to have a large effect size such as premutations in the FMR1 gene and mutations in the BRCA genes [37–42]. Additionally, in families with a history of premature ovarian insufficiency with a known genetic basis, testing of the target genes may be of value.
X Chromosome Abnormalities
Normal ovarian development requires the presence of two functional X chromosomes. From 8.8% to 32% of women with premature ovarian insufficiency are found to have chromosomal abnormalities, mostly involving the X chromosome (e.g., monosomy X (Turner syndrome), trisomy X, X chromosome deletions and translocations, and X-isochromosomes. A study of Chinese women with premature ovarian insufficiency found that 12.1% had X chromosome structural abnormalities [34].
Array comparative genomic hybridization disclosed smaller X chromosome duplications and deletions in women with premature ovarian insufficiency. X chromosome terminal deletions (Xp11 to Xp22.1) are associated with primary amenorrhea, and deletions from Xq13 to Xq27 are associated with primary amenorrhea or primary ovarian insufficiency.
As noted earlier, premutations in the FMR1 gene (fragile X mental retardation gene) are associated with non-syndromic premature ovarian insufficiency and poor outcomes in infertility treatment. The FMR1 premutation is an expansion (56 to 200 copies) of a cytosine-guanine-guanine (CGG) repeat in the 5′-untranslated region, which reduces FMR1 mRNA translation. Located on the X chromosome (Xq27.3), FMR1 encodes an RNA-binding protein, FMRP, that controls mRNA stability. Population studies suggest that the number of CGG repeats is not linearly correlated with the extent of ovarian dysfunction. A mouse model suggests that the accumulation of the premutation RNA causes a reduction in the number of growing follicles. About 20% of women carrying the FMR1 premutation have primary ovarian insufficiency, and 0.8% to 7.5% of women with sporadic premature ovarian insufficiency have a FMR1 premutation.
Germline Mutations in BRCA1/BRAC2
Women with germline mutations in the BRCA1 and BRCA2 genes, which are involved in double-strand DNA break repair, have reduced ovarian reserve as assessed by biomarkers (lower AMH levels) and lower primordial follicular densities provide evidence for the pivotal role of DNA repair mechanisms in maintaining the germ cell compliment [37–40]. Germline BRCA1 mutations are also associated with increased double-strand DNA breaks in primordial follicle oocytes, and a lower number of mature oocytes recovered after ovarian stimulation [36–42]. BRCA1/BRCA2 mutations vary among populations (more prevalent in Ashkenazi Jews, lower in Latinx, African-Americans, and Asian-Americans).
With the advent of broader access to genetic testing for germline mutations associated with breast and ovarian cancers, more women are obtaining information on the status of their BRCA1/BRCA2 genes as well as other genes implicated in breast and ovarian cancer, which include genes that have been associated with age of natural menopause. Based on the associations described earlier between BRCA genes and ovarian function, clinicians have been encouraged to tell women with germline BRCA1 mutations not to delay pregnancy [42].
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