Abstract
According to the most recent cancer statistics, more than 870,000 new diagnosis of cancer are expected in the US female population in 2018, with the three most common cancers in women being breast, lung, and colorectal cancers [1].
Several improvements have been made in the early diagnosis and treatment of infant and adults cancer and these advances have resulted in greatly increased life expectancy and chances of survival. Nevertheless, some oncological treatments, although leading to cancer cure rates higher than 90%, have a detrimental effect in the reproductive potential of children and young women, resulting in a population at high-risk of developing premature ovarian insufficiency (POI) and therefore infertility [2].
In order to prevent the risk of facing this outcome, fertility preservation options are offered to these patients in order to protect their fertility potential prior to gonadotoxic treatment. Among the available options, ovarian tissue cryopreservation and transplantation is the only method suitable for prepubertal girls and adult women who require urgent treatment.
Introduction
With the advent of culture models for studying the process of ovarian folliculogenesis some 40 years ago, opportunities arose for the more systematic evaluation of the factors that regulated ovarian function [1]. The initial focus of studies using cultured follicles emphasized two of the then widely recognized roles of the follicle in mammals: the production of ovarian steroid hormones and of viable oocytes during the process of ovulation. As our understanding of the molecular and cellular complexity of this tissue compartment has evolved and deepened, so too has the need to redefine the major functions of the follicle at both local ovarian and systemic levels in the context of reproduction in mammalian species, especially as it relates to the origins and treatments for human infertility [2]. Thus, a shift in the motivation to use cultured follicles in humans has taken place owing primarily to the rapidly evolving field of fertility preservation. Through an interesting turn of events dictated by the need to maintain and propagate human oocytes that would be capable of supporting term gestations, a dire need has been recognized that would enable optimization of follicle functions under in vitro conditions in order to realize ovarian capacity for young women who have had their fecundity seriously compromised as a result of genetic, environmental or iatrogenic life-sparing treatments such as those involved with the management of cancer [3–5].
The introduction of technology that permits the cryopreservation of ovarian tissues has opened the prospect of sustaining and storing primordial follicles from individuals that could at a later time be thawed and subjected to prolonged culture. Exactly what conditions will be required to sustain follicular function to support the growth phase of oogenesis has yet to be worked out, as are the factors that would normally be involved in this protracted and oocentric phase of folliculogenesis. Working out conditions that could recapitulate the structural, molecular and cellular properties of the follicle in vitro remains a major challenge in the area of fertility preservation, as does the development and implementation of novel technologies that would permit a reproducible and reliable assessment of oocyte integrity. It is the intent of this chapter to review methods that have been in practice for the evaluation of follicle culture integrity and to point out the strengths and deficiencies of these methods. Finally, the techniques that loom on the horizon which could meet the criteria necessary for monitoring follicular integrity will be considered, as new sentinels or biomarkers could predict the developmental capacity of oocytes for the field of fertility preservation.
While direct measures of oocyte developmental competence remain a lofty goal, more recent efforts to characterize this aspect of follicular integrity have tended to rely upon indirect ways to avoid unnecessary damage to the oocyte that might already have been compromised by ex vivo conditions and/or cryopreservation [3, 6, 7]. For this reason, we propose a viewpoint of follicle integrity that emphasizes the heterocellular character, which includes the somatic granulosa and theca elements and offers multiple parameters for assessment that in the larger picture may better represent the overall quality of the enclosed oocyte [8]. This viewpoint is further supported by the recent appreciation of the level of signaling and metabolic integration that reflects the continuity of both negative and positive feedback mechanisms between the oocyte, granulosa and theca cells that comprise the ovarian follicle [2]. While this perspective excludes formal contributions from the surrounding stromal compartment within which the follicle develops, the role of the stroma cannot be overlooked with respect to the contributions that it makes during the course of in vivo folliculogenesis in relation to thecal lineages including the microvasculature of antral stage follicles. Thus, what follows is a summary of the current and future biomarkers that may be useful in evaluating the integrity of cultured follicles. We begin with somatic components of the follicle and finish with those assayable properties of cultured oocytes that must be analyzed before the use of oocytes in assisted reproductive technology (ART) applications such as in vitro fertilization (IVF), embryo culture and transfer.
Evaluating Somatic Cell Components of the Follicle
The traditional array of biomarkers for the ovarian follicle has emphasized endocrine performance, especially as related to the biosynthesis and secretion of estradiol [9, 10]. Built on the classical two-cell model, assays that monitor the production of thecal androgens in response to luteinizing hormone (LH) and granulosa cell-derived estradiol in response to follicle stimulating hormone (FSH) remain the mainstay for evaluating follicular integrity under in vivo and in vitro contexts [8]. It can be argued, however, that these valid and predictive biomarkers for the endocrine health of the follicle overshadow the most relevant attribute that directly bears on the overall developmental status and health of the oocyte (Figure 28.1).
Figure 28.1 Diagram illustrating the gradient of influences from the oocyte to granulosa and thecal compartments of the ovarian follicle that serve as guideposts for monitoring the cellular and molecular integrity of follicles maintained in culture. Note that the primary function of oocyte secreted factors such as growth differentiation factor-9 (GDF-9) is to prevent the proliferation (hyperplasia) and differentiation (hypertrophy) of the steroidogenic functions of the follicle prior to and following ovulation. Thus, signs of steroidogenesis in culture are likely to reflect a loss in this command function of the oocyte resulting in impaired viability and developmental competence
Thus, the major phases of oogenesis, during which both oocyte growth and preparations for the completion of meiosis and fertilization occur, occupy the earliest stages of follicle development between the activation of the primordial stage and entry into the secondary or antral stage when estrogen production commences upon the induction of aromatase activity in response to FSH [11–14]. That the latter property appears commensurate with the rapid expansion of the theca and granulosa by a burst of cell proliferation is often identified as a measure of follicular health, but the essential question is how do these FSH-induced attributes of the follicle, increased proliferation and aromatase activity, influence the terminal stages of oogenesis if the goal of culturing follicles is to obtain high-quality oocytes?
There can be no question that the measure of any organ or tissue culture system is the health and well-being of its constituent cells. Moreover, as in many other developmental systems that are highly regulated in a stage-specific fashion, the ovarian follicle engages the basic cellular properties of differentiation, proliferation, survival and death, and each of these apply to both the theca and granulosa at select stages of follicular development (Table 28.1).
Table 28.1 Summary of major functional properties for ovarian follicles that have been used in the assessment of tissue integritya
| Property | Follicle stage | Biomarker | Vital |
|---|---|---|---|
| Quiescence | Primordial | Chromatin/PTEN | Yes (Hoechst 33342) |
| Proliferation | Primary, secondary | BrdU, PH3, MPM2 | Yes (Click-IT) |
| Apoptosis | Secondary, antral | TUNEL, caspase 3 | Yes (Hoechst 33342) |
| Autophagy | Primordial (? others) | Beclin/ATG | Yes (LysoTracker Red, acridine orange) |
| Differentiation | Antral | Aromatase, LHr | No |
a Note that many of the biomarkers employed to date draw on the use of immunocytochemical and histological assays requiring tissue destruction. Also, not all markers are pertinent to all stages of folliculogenesis. Some examples of vital biomarkers that are under development for determining cultured follicle integrity are shown in the last column.
It is not surprising then that the most commonly used measure of cultured follicle integrity is hypertrophy over time, whether follicles are cultured under adherent two-dimensional conditions or within matrices of various kinds that retain a three-dimensional architecture [15–20]. While standard protocols deploying assays for cell proliferation (3H-thymidine, BrdU incorporation or cell cycle markers such as phosphohistone-3, PCNA, etc.) or apoptosis (TUNEL, caspase-3) offer postscripts for the relative fraction of viable cells within a follicle (Table 28.1), in the end these are crude and retrospective assays that add little to the immediate needs of the clinician requiring a more real-time assessment of follicle integrity. Toward this end, several new probes have gained usage in the evaluation of tissue culture models that take advantage of the speed, sensitivity and spectral properties of microplate readers. This new generation of reagents permits resolution of metabolic activity, including reactive oxygen generation, cell proliferation and even identification of rapid-versus-slowly dividing cells within an organ or tissue culture using multi-well formats, which should avail the optimization of conditions that support oogenesis. Moreover, as discussed subsequently, the link between DNA damage sensing and repair is fast becoming a major determinant in the assessment of follicle integrity as it relates to both somatic and germ cell components, and these assays have introduced a range of sensitivity and precision that will materially advance the field of follicle culture.
As with most in vitro systems, culture environments create adverse conditions that are known to affect DNA integrity, often due to the generation of free radicals in response to high oxygen tension [21]. Given these deleterious side effects of culture environments, genomic integrity is one area of follicle evaluation that has received little attention and requires closer inspection. Many new reagents are available for evaluating the cascade of events associated with the detection of DNA damage, as well as the activation and completion of the DNA-repair pathway that should be active in both somatic and germ cells of the follicle. We have recently been exploring the components of this pathway in the mammalian ovary and find that a variety of insults – ranging from advancing maternal age, reactive oxygen species (ROS) generation during culture and exposure to chemotherapy agents such as cyclophosphamide – bring about rapid and reversible changes in the degree of DNA damage and repair in ovarian cells. As shown in Figure 28.2, granulosa cells isolated from bovine ovarian follicles provide a useful culture system for evaluating DNA damage by immunofluorescence microscopy.
Figure 28.2 Range of DNA double-strand breaks detected in bovine granulosa cell cultures observed before or after exposure to cyclophosphamide. Top row (a–e) illustrates staining profiles after labeling with gamma-H2AX antibody that reveals solitary foci (a), multiple foci (b) and various patterns (c–e) that presumably reflect earlier stages in DNA lesioning prior to foci formation as indicative of active sites of DNA repair. Bottom row (a1–e1) demonstrates total chromatin. Scale bar represents 10 µm
A wide variety of reagents is now available that detects epitopes that appear in response to spontaneous or induced double-strand breaks. Among these, antibodies that recognize histone modifications that occur at the site of strand breaks reveal both the extent and magnitude of lesions in the form of foci of varying number and size within single cell nuclei. By fixing cells in response at various times following exposure to the chemotherapy agent cyclophosphamide, these foci appear in increasing numbers that can be quantified as the mean density/nucleus. This gives us an understanding of the relative time course of the induction of DNA damage and the rate of repair upon drug removal or after altering culture conditions (Figure 28.3).
Figure 28.3 Bovine granulosa cells cultured in the absence (a–c) or presence (d–i) of cyclophosphamide for 4 h (d–f) or 24 h (g–i) that have been fixed and stained for the demonstration of total chromatin (a, d, g), gamma-H2AX (b, e, h) and RAD51 (c, f, i). With progressive repair, as evidenced by the formation of discrete gamma-H2AX positive nuclear foci, cytoplasmic RAD 51 assumes an intranuclear location coincident with the sites of DNA double-strand breaks. Scale bar represents 10 µm
When used in combination with reagents that detect components of the repair complex, such as RAD51 or BRCA1, further insight into the DNA repair capacity can be obtained. This approach is applicable to sections of ovarian tissue or cultured follicles, and thus adds an important dimension to the assessment of DNA integrity in both somatic and germ line components of the follicle under in vivo or in vitro conditions.
Center Stage: Supporting and Maintaining Oogenesis
In the context of fertility preservation, the singular objective of follicle culture is to provide an ex vivo context within which the growth and maturative stages of oogenesis can proceed unabated and without inflicting damage to the oocyte as it acquires the capacity to support embryonic development [18, 22, 23]. As alluded to earlier, to a certain extent a paradox emerges, since the most valid predictors of the success of follicle culture will reside in the ability to document that indeed oocyte quality has been established and maintained for whatever duration of follicle culture is needed to achieve this objective [5, 24–26]. Thus, while a substantial body of evidence supports the idea that folliculogenesis consumes upward of 100 days in humans [1, 2, 8], we remain ignorant as to the exact chronology and duration of events that are required to support the entire process of oogenesis as it occurs within the confines of the follicle. Moreover, it remains unclear as to whether the process of folliculogenesis reflects a developmental continuum or one that is punctuated by episodes that attend to key steps in oogenesis dictated intrinsically or from cyclic variations in gonadotropin availability or expression of receptors for LH or FSH [27–30]. Answers to these questions will inform future approaches in this field in conjunction with the emergence of assays that more directly target and report upon the quality of the oocyte within cultured follicles.
As noted earlier, short of assessing the ability of oocytes to complete meiosis, engage successfully in fertilization in vitro and support preimplantation development [1, 7, 31], there are few biomarkers that could safely be considered as reliable or efficient in determining oocyte quality.
Those that have been proposed – such as metabolic substrates, visual indicators that rely on the use of fluorescent reporters or other vital indicating dyes (Table 28.1) – pose limitations pertaining to assay sensitivity, free radical generation and metabolic perturbations, respectively, thus precluding their general utility. For these reasons, non-invasive and non-perturbing optical assays remain a cornerstone for the assessment of oocyte quality in materials that will be subjected to IVF and embryo culture as a measure of developmental competence.
The more promising indirect biomarkers are those that monitor the integrity of the interaction between oocytes and granulosa cells, whereby both the differentiative state of the oocyte and its surrounding granulosa cells can be assayed [31, 32].
Based on the tenet that establishing contact between the oocyte and granulosa is realized through the anchoring of transzonal projections (TZPs) at early stages of follicluogenesis, the effectiveness of these connections is likely to be a direct reflection of the metabolic synergy required to acquire meiotic competence and preserve the oocyte in a developmentally competent state for the duration of culture [27, 33]. Earlier studies using mouse ovarian follicles emphasized the importance of minimizing FSH exposure as this resulted in the precocious resumption of meiosis, a consequence that would materially exacerbate the problem of oocyte aging [17]. Thus, more recent efforts to achieve maintenance of oocyte health during oocyte culture have viewed the inclusion of FSH at inappropriate times to be hazardous to the oocyte while advancing follicle differentiation to the next level [1, 14, 18, 30]. Interestingly, these adverse effects of FSH appear to be due to a direct action on the TZPs as they are rapidly and irreversibly retracted and may delimit the availability of nutrients and/or signals that operate during both the acquisition of meiotic competence and maintenance of meiotic arrest [34]. It may well be that, besides identifying more suitable media conditions favoring oocyte viability, other factors will be discovered that effect a more stable interaction between oocytes and granulosa cells based upon the composition of the extracellular matrix [6, 10] and whether or not cultures are maintained in a two- or three-dimensional context [18–20, 35]. One final factor that is likely to contribute to the stability of TZPs is growth differentiation factor-9 (GDF-9). Originally discovered as a critical oocyte produced factor required for the development of secondary follicles [36], GDF-9 was shown to be essential for the establishment of TZPs in mouse knockout models [37]. Whether exogenous recombinant forms of GDF-9 or related oocyte signaling molecules will facilitate metabolic synergy remains to be demonstrated, but this variation in culture conditions would be worth exploring in seeking alternative strategies that would preserve the native architecture of the follicle in a fashion consonant with support of oocyte development and metabolism [38].
Maintaining cellular interactions within the follicle is relevant to the effects of ovarian tissue cryopreservation or primordial follicle organization. The hyperosmotic stress induced by cryoprotectants in both slow freeze and vitrification protocols may disrupt cell interactions in distinct ways. As shown in Figure 28.4, slow freeze protocols tend to perturb the stromal–basement membrane interface whereas vitrification impacts the interface between the oocyte and surrounding granulosa cells.
