Ultrastructural analysis of human immature oocytes retrieved after two different protocols of controlled ovarian hyperstimulation

Summary The study presented in this PhD dissertation thesis was performed at the: 1) Dept. of Life, Health and Environmental Sciences of the University of L’Aquila and, 2) Dept. of Anatomy, Histology, Forensic Medicine and Orthopaedics, Sapienza University, Rome, Italy. Introduction Immature oocytes can be an alternative source of oocytes for Assisted Reproductive Technologies (ARTs). These oocytes can be retrieved by patients undergoing different controlled ovarian hyperstimulation protocols (COH), such as the conventional “full” (fCOH) or the less aggressive “mild” (mCOH) protocols. These, so-called “leftover” oocytes can increase the yield of total available oocytes, especially for poor responder patients or those having an unsynchronized cohort of follicles. The present PhD thesis aimed to evaluate the effects of two different COH protocols, a GnRH agonist (fCOH) stimulation and a GnRH antagonist (mCOH) stimulation, on the fine structure of fresh human immature GV-stage oocytes, to consider the ultrastructure of well-preserved organelles as indicator of GV quality preservation. Methods Fresh GVs were retrieved from consenting donor women undergoing ARTs due to tubal or male infertility factors. COH was achieved by: 1) a standard long protocol, with GnRH agonists (fCOH protocol) or 2) a mild protocol, with GnRH antagonists (mCOH protocol). After retrieval, oocytes were washed in phosphate buffered saline (PBS) solution, fixed in 2.5% glutaraldehyde/PBS and subjected to standard preparative for light (LM) and transmission electron microscopy (TEM). The following parameters were evaluated by LM and TEM and taken into consideration for qualitative assessment of the ultrastructural preservation of oocytes: general features (including oocyte shape and dimensions); morphology of the nucleus, nucleolus, chromatin and nuclear envelope (including nuclear membrane integrity); microtopography, type and quality of the organelles; mitochondria shape, number and dimensions; presence and extent of ooplasmic vacuolization; position, number and dimensions of cortical granules; integrity of the oolemma and microvillus pattern; appearance of the perivitelline space (width, presence of fragments); zona pellucida texture. Results Most of fCOH and mCOH oocytes were well preserved and showed a roundish and centrally located nucleus. The nucleolus was reticulated and connected with dense heterochromatin spots in both oocyte groups. Mitochondria were usually located in a perinuclear position, sometimes associated to small vesicles; in the mCOH group, they showed a significant increase in the number, respect to fCOH. Numerous electron negative and membrane-bounded vacuoles were located close to the nucleus in both groups, but they appeared significantly larger in fCOH than in mCOH oocytes. Cortical granules were located both in the subplasmalemmal area and dispersed throughout the whole ooplasm, even if less abundant; the number of cortical granules was significantly higher in mCOH oocytes, compared to the fCOH ones. An intact and continuous zona pellucida were present in both groups; microvilli were uniformly distributed in both groups, with some areas of rarefactions in fCOH oocytes. Conclusion The data of the present study showed organelle-specific alterations in the oocytes observed, probably related to the applied COH protocol. In particular, the significantly lower number of mitochondria and the biggest size of vacuoles in fCOH oocytes TEM: transmission electron microscopy, respect to what observed in mCOH oocytes, may suggest that some microdomains of the GV-stage oocytes may be affected by the hormonal stimulation.