Development Before to Fertilization: Oocyte-Specific Factors
Oocyte Maturation Blocks
Single or multiple defect(s) in the female gamete may be apparent before fertilization. Oocytes undergo a precise and multifaceted developmental program during which each gamete acquires the ability to support the creation of a new zygote and its development to term. Although most retrieved oocytes in a cohort have typically progressed to metaphase II (MII), some oocytes may be arrested at various points at or beyond the arrested germinal-vesicle (GV) stage. Moreover, several studies have reported on recurrent oocyte immaturity across entire retrieved cohorts for a patient. Such observations have been made even when full growth of oocytes has occurred, making it unlikely that a deficiency in meiotic competence acquisition occurred during folliculogenesis. Of particular relevance to this review are repeated cases attributed to oocyte-inherent defects for which alterations in stimulation protocols, timing of retrieval, and subsequent rescue in vitro maturation (IVM) failed to solve the meiotic arrest. Oocyte maturation block cases discussed here are ones for which defects in external factors are not suspected.
The existence of complete meiotic arrest may not be surprising given the complex control and multiple arrest points that characterize normal female meiosis. A heightened susceptibility for errors may come with such complexity. The exact prevalence cannot be stated because cases are not necessarily documented, and when reported, they are typically presented as case studies rather than as an incidence. A survey of all oocyte maturation block cases published to date (up until July 2011) revealed a total of 30 patients, with 4, 17, and 9 patients afflicted with GV, metaphase I (MI), and mixed (GV and MI) arrest, respectively.
To assist in the management of these maturation arrest cases, it is informative for the laboratory to report the exact morphological defect, such as at least the presence of a GV nucleus or absence of a polar body in all retrieved oocytes. A uniform abnormality across an oocyte cohort is rare, thus raising suspicion for the success of subsequent cycles. Ideally, diagnostic measures can then be taken to characterize the defect further. Approaches to date have included immunofluorescence and confocal laser microscopy on fixed oocytes or live polarized microscopy for evaluation of the chromatin and cytoskeleton, ultrastructural, and cytogenetic analyses. The resulting information enabled the identification of exact arrest points or defects in the cytoskeleton, such as the absence of spindles, normal or abnormal bipolar spindles depending on the patient, gross aberrations in spindle organization, or failure of first polar body extrusion despite meiosis-I nuclear division. Diagnostic efforts to date have already demonstrated a spectrum of cellular abnormalities despite a uniform arrest at MI based on morphological assessment alone. With different phenotypes, there are likely multiple etiologies underlying the total immaturity of the oocyte cohort.
A limitation of many studies to date includes the analyses of oocytes after prolonged culture. In vitro oocyte aging is a phenomenon known to alter cytoskeletal architecture, and results may be confounded due to delayed analysis, although laboratories have strived to diminish or eliminate this issue altogether. In addition, a single arrest point is assessed but it may not represent the first, or even all, potential defect(s) in oocyte development. For instance, a defect originating at one developmental milestone may not manifest until a later event.
New diagnostic approaches should continually be explored, particularly as technological advances proceed and our understanding of gamete development increases. The documentation of family history may well be informative; indeed, a study of two sisters with a meiosis-I total arrest suggested the inheritance of an autosomal recessive trait at the root of the oocyte factor for infertility. A genetic basis would not be surprising given the several mouse mutations in key regulatory genes that exhibit anomalies in meiotic progression. Defects in events seminal to meiotic resumption, such as maturation-promoting factor activation and/or a decrease in cyclic adenosine monophosphate levels, may explain a complete GV arrest. Similarly, MI arrest may result from cell-signaling issues (e.g., anaphase-promoting complex/cyclosome) or aberrations in the spindle assembly and chromosome alignment checkpoints (e.g., spindle assembly checkpoint). Lastly, several cases document a mixed phenotype with GV and MI arrests, perhaps reflecting insufficient compensatory mechanisms.
Taken together, there is a growing list of candidate genes characterized in animal models, and this knowledge promises to inform our ability to diagnose the exact molecular defect(s) underlying the various cases of oocyte maturation arrest in humans. Of course, diagnostic efforts should also consider other potential issues in folliculogenesis and/or cumulus cell-oocyte interactions.
If diagnostic approaches indicate a major oocyte defect, the best route of management then becomes counseling the patient toward oocyte donation or adoption. Early diagnosis is obviously beneficial because it would prompt the patient to proceed with one of these alternatives sooner rather than later, not after multiple stressful and costly unsuccessful cycles.
Also supporting the benefits of early intervention is a case report of a patient with mixed GV and MI arrest; as early as cycle 2, in vitro culture allowed the MI oocytes to extrude a polar body by 12 hours post-retrieval, followed by intracytoplasmic sperm injection (ICSI), which then resulted in fertilization, the transfer of a developmentally competent embryo, and the birth of a healthy singleton. However, most cases fail to show the benefits of rescue IVM. Of note is the limitation that to date, all rescue IVM attempts for oocyte maturation block have been performed on cumulus-free oocytes denuded for ICSI. Further, the use of unstimulated or mildly stimulated IVM cycles did not rescue cases of GV or MI arrests.
Due to the current limitations of IVM, therapies should thus be envisaged to help patients diagnosed with recurrent meiotic arrest; one approach recently explored the use of an artificial activation protocol. Although it was hypothesized that artificial activation with a calcium ionophore (i.e., a compound that induces an intracellular calcium increase) would stimulate MI arrested oocytes to resume meiosis, the therapeutic approach proved grossly unsuccessful. First are needed not only future evaluations of the exact molecular deficiency in these disparate cases of MI arrest but also optimal strategies for triggering meiotic progression. Although the protocol put forth by Heindryckx et al was founded on testing by others in a rodent model, the approach is extremely experimental and not yet warranted for clinical use. Importantly, the field must exercise great caution when using therapeutic strategies to coerce oocytes to progress to MII, particularly given the reported severe aberrations in spindle organization. We cannot risk fertilizing oocytes that may be intrinsically prone to result in aneuploid gametes and embryos. Only the future will unravel whether targeted and safe therapeutics can be developed to reverse the failure of oocytes to mature.
Oocyte Dysmorphisms
Other than meiotic arrest, there may be gross abnormalities in the morphology of the oocyte. For instance, very large vacuoles or smooth endoplasmic reticulum clusters (sERCs) were reported in a patient with poor fertilization and embryonic arrest. Another case report presented the recurring incidence of smooth endoplasmic reticulum aggregates, and of concern was the incidence of major fetal anomalies from all conceptions. Documenting such unique patient cases thus proves informative in exercising caution when transferring embryos from an oocyte cohort with this type of abnormality. With respect to management using the patient's own oocytes, only invasive procedures may help alleviate the defect. Removing or aspirating the vacuoles with micromanipulation may provide a solution, but they raise serious safety concerns that unfortunately cannot be easily tested in these rare clinical cases and without an appropriate animal model that recapitulates the unique oocyte phenotype.
Empty Follicle Syndrome
Also under the loose diagnosis of "egg factor" is empty follicle syndrome (EFS). Genuine EFS is defined as no oocytes retrieved despite follicular development and a response to ovarian stimulation that appears normal. In recurrent cases of genuine EFS, it has been hypothesized that the suspected cause for EFS lies in aberrations in follicular development, likely in late stages, resulting in oocyte loss. One solution in such cases may be to retrieve immature oocytes before the demise of the follicle. Indeed, this approach has proved successful, with in vitro matured oocytes resulting in viable embryos.