Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

BACKGROUND: The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). METHODS: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. RESULTS: An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. CONCLUSION: These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Changing clinical significance of oocyte maturity grades with advancing female age advances precision medicine in IVF.

In current IVF practice, metaphase-2 (M2) oocytes are considered most efficient in producing good quality embryos. Maximizing their number at all ages is standard clinical practice, while immature germinal vesicle (GV) oocytes are mostly automatically discarded. We present preliminary evidence that oocyte maturity grades with advancing age significantly change in their abilities to produce good quality embryos, with M2 oocytes significantly declining, GV oocytes improving, and M1 oocytes staying the same. These data contradict the over-40-year-old dogma that oocyte grades functionally do not change with advancing age, supporting potential changes to current IVF practice: (1) Stimulation protocols and timing of oocyte retrieval can be adjusted to a patient's age and ovarian function. (2) In older and younger women with prematurely aging ovaries, GV oocytes may no longer be automatically discarded. (3) In some infertile women, rescue in vitro maturation of immature oocytes may delay the need for third-party egg donation.

Sperm centriolar factors and genetic defects that can predict pregnancy.

The human sperm centrosome, comprising the two morphologically distinct centrioles and associated pericentriolar materials, plays a crucial role in fertilization and early embryonic development after fertilization. Once inside the oocyte, the sperm centrosome serves as a microtubule-organizing center, orchestrating mitotic spindle formation, chromosome segregation, and syngamy. Abnormalities of the sperm centrosome can lead to abnormal embryonic development and embryonic chromosomal instability, and are associated with pregnancy loss. Recent research has shed light on the molecular composition, regulation, and function of this vital organelle. Understanding the intricacies of the sperm centrosome is crucial for elucidating the mechanisms underlying successful fertilization and early embryonic development, as well as addressing infertility and developmental disorders associated with centrosomal defects.