In Vitro Activation: A Dip Into the Primordial Follicle Pool? Free

Current practice of assisted reproduction requires three essential elements: mature female and male gametes, fertilization, and embryo transfer. From 1965 to 1992, fertilization was the focus of in vitro fertilization (IVF). The origins of IVF can be traced to 6 weeks in the summer of 1965 when the British scientist Robert Edwards traveled to the United States in search of a source of human oocytes for his studies. Edwards began work in the laboratory of Drs. Howard and Georgeanna Jones at Johns Hopkins and, having established the time required for a human oocyte to complete metaphase II (1), proceeded to fertilize the first human oocyte in vitro (2). Edwards returned to the United Kingdom in 1965, but it was not until 1978 that he and Patrick Steptoe announced the birth of Louise Brown (3), the first IVF baby; an achievement that launched the field of assisted reproduction and led to Edwards receiving the Nobel Prize in 2010. The number of male gametes remained a limiting factor, however, until Palermo, Joris, Devroey, and Van Steirteghem achieved fertilization of an oocyte by injection of a single sperm in 1991–1992 (4), thus inventing the technique of intracytoplasmic sperm injection (ICSI).

With the ability to achieve fertilization for couples having severely limited numbers of male gametes, the crucial limitation of assisted reproduction has become the number of available female gametes. In human females, proliferation of primary germ cells is maximal in utero. Current understanding is that the germ cells arrest at prophase I and become surrounded by a thin ring of granulosa cells to become primordial follicles. Estimates are that there are 2 million primordial follicles at birth, with 400 000 remaining by puberty, and although some are ovulated, most undergo atresia, so that approximately 1000 follicles remain at menopause (5). Thus, there is a natural decline in oocyte number with female age. In addition to oocyte number, oocyte quality also declines naturally with female age, as has been aptly demonstrated by population studies (6). The age-related reduction in female fertility is oocyte-dependent and is not attributable to aging of the female reproductive axis, as illustrated by the fact that women 45 years of age have no reduction in live births if donor oocytes are used (7). A clinically significant reduction in the pool of primordial follicles is known as “diminished ovarian reserve” (DOR), and the condition affects up to 10% of couples pursuing assisted reproduction. Even more significant reductions in oocyte numbers are encountered in women with primary ovarian insufficiency (POI; also known as premature ovarian failure, or POF).

An important point is that with DOR, and very often with POI, the ovary contains residual dormant primordial follicles that theoretically could be activated to become mature oocytes. Furthermore, based on current evidence from women who eventually developed POI (8) or had POI in remission (9, 10), offspring from women with POI were normal. Similarly, offspring from women with DOR who achieve pregnancy are also normal. These two observations suggest that “dipping” into the dormant primordial follicle pool is likely to result in normal offspring. The limited availability of female gametes has focused attention on primordial follicle dormancy and the steps leading to activation of the primordial follicles, which remain poorly understood. The question is: how can primordial follicles be activated?

Several recent reports suggest that it may be possible to activate dormant primordial follicles using a combination of mechanical signaling and biochemical factors. This new method is called “in vitro activation,” or IVA (11). Evidence that mechanical signaling could promote activation of primordial follicles was first explored in a murine model by Cheng et al (12). The study indicated that mechanical disruption of ovarian structure and polymerization of actin led to inhibition of the Hippo pathway and oocyte growth (reviewed in Ref. 13). Based on the techniques established in mice, in 2013 Kawamura and Hsueh reported on a woman with POI who gave birth (11). Since that report, several other births have been reported by the same group (14), and two other live births have been reported from medical centers in China and Spain (cited in Ref. 15). These outcomes and the report by Zhai et al (16) support the clinical utility of the IVA technique.

Before the reports of IVA, there was considerable evidence that mechanical signaling plays an important role in oocyte maturation. Work from the Evans laboratory and others emphasized the importance of mechanical signaling for meiosis (17). Additionally, studies of Woodruff and Shea have revealed a crucial importance of mechanical signaling for follicle development (18) and primordial follicle survival in ovarian transplantation (19). Likewise, a recent report from Oktay’s group showed that scaffolds increased the survival of transplanted ovarian tissue (20). Furthermore, recent measurement of oocyte tension has been shown to correlate with outcome at assisted reproduction (21). Collectively, these findings, in addition to the work of other groups, provide compelling evidence that mechanical signaling is critical for proper oocyte development. Indirectly, these observations suggest that mechanical signaling may contribute to oocyte “storage” and dormancy of primordial follicles. Thus, several lines of evidence support the hypothesis that altering mechanical signaling might represent an approach to promote activation of follicle growth.

To be sure, some reports have questioned some of the mechanisms supporting IVA. One study suggested that YAP, a transcriptional coactivator in the Hippo pathway, was not involved in oocyte development (22). Also, it is clear that in some cases, ovaries of women with POI do not contain any dormant primordial follicles; thus, IVA will not work for all women. Additionally, the length of time after development of POI could be an important factor; it is possible that success with IVA may be limited to recent-onset POI. Likely, as experience with the technique improves, IVA may be more effective for specific cases of impaired ovarian function, but not for others. Moreover, the technical and surgical methods for the procedure have already been revised (reviewed in Ref. 23), and it is likely that further refinements might augment success.

Although there have been several reports of ongoing folliculogenesis in adult ovaries arising from oogonial stem cells (24, 25), these reports have been controversial, and other groups using different approaches have not been able to substantiate the phenomenon (26, 27) or questioned the key reagents used (28, 29). Other work has centered on augmenting primordial follicle formation, which requires migration of precursor granulosa cells around individual oocytes. Primordial follicle formation is facilitated by Ras-related C3 botulinum toxin substrate 1, which causes nuclear trafficking of signal transducer and activator of transcription 3 promoting transcription of several oocyte-specific proteins. These oocyte-derived proteins activate receptors on pregranulosa cells to induce translation of neurogenic locus notch homolog protein 2, which promotes primordial follicle formation (30). Although the prevailing opinion of most reproductive scientists is that, if it occurs, neo-oogenesis and resulting primordial follicle formation contribute very little to the primordial follicle pool in the adult, it is important to point out that IVA does not appear to involve neo-oogenesis. Rather, IVA leads to activation of existing dormant primordial follicles.

At present, few pregnancies with IVA have been reported. However, if IVA is substantiated with larger studies, the ability to access the pool of dormant primordial follicles could offer new hope to millions of women with diminished ovarian reserve, whether age-related (physiological) DOR or non-age-related (pathological) DOR (31). In many countries, childbearing has been delayed as women pursue careers and use contraception. An unwanted consequence of postponing pregnancy is the natural decline in fertility, which results in age-related DOR in women desiring pregnancy. IVA could represent a solution to that conundrum. Understanding that the mechanical stiffness of transplanted ovarian tissue may increase primordial follicle survival has ramifications for fertility preservation in women with cancer (14, 20). Furthermore, based on the principles of mechanical and biochemical activation, more effective preservation and activation of autotransplanted tissue might be possible.

In conclusion, if substantiated by future clinical studies, IVA could represent a paradigm shift for fertility care. Although current evidence is very limited, if future experiments substantiate effectiveness of the technique, IVA could prove as revolutionary to the practice of assisted reproduction as IVF or ICSI.

Acknowledgments

J.H.S., K.C., and O.Y. are supported by the Howard and Georgeanna Jones Endowment.

Disclosure Summary: The authors have nothing to disclose.

Abbreviations

 
• DOR

  diminished ovarian reserve

 
• ICSI

  intracytoplasmic sperm injection

 
• IVA

  in vitro activation

 
• IVF

  in vitro fertilization

 
• POI

  primary ovarian insufficiency.

References