A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Summary

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Abstract

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 °C and 5% CO₂. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Introduction

Female infertility is a pathology that affects an increasing number of women. According to the World Health Organization, around 20% of couples are infertile, with a 40% due to female infertility. In addition, one third of women undergoing cancer treatments (300,000/year and 30,000/year in the USA or Italy, respectively) develop premature ovarian failure.

A strategy to prevent infertility in cancer patients is the isolation and cryopreservation of ovarian follicles before the oncological treatment, followed by in vitro maturation (IVM) of GV oocytes to the MII stage (GV-to-MII transition). The availability of non-invasive markers o....

Protocol

All procedures involving animals were approved by the Institutional Animal Care and Use and Ethical Committees at University of Pavia. Animals were maintained under conditions of 22 °C, 60% air humidity and a light/dark cycle of 12:12 h.

1.Ovary Isolation

1. Inject intraperitoneally 2 four-to-eleven week-old CD1 female mice with 10 U of follicle stimulating hormone with a sterile 1 mL insulin syringe.
2. Wait 46-48 h.
3. Weigh the mouse and anesthetize with an intramuscular injection of 50 mg/kg of Zoletil (Tiletamina and Zolazepan cloridrate). Euthanize by cervical dislocation.
4. Grasp the skin covering ....

Results

Figure 2 shows a representative developmentally competent and incompetent oocyte, respectively at the beginning (GV) and at the end (MII) of the IVM procedure. IVM of fully-grown mouse oocytes occurs during 15 h culture. The Time-Lapse observation records the progression of meiosis and detects major meiotic events, including the GVBD and the extrusion of the first polar body.

The analysis and compar.......

Discussion

There are several critical steps one should take care of while performing this protocol with mouse oocytes as well as with those of other species. Once isolated from their follicles, oocytes should be immediately transferred into the recording drops, as the separation from the companion cumulus cells triggers the beginning of the GV-to-MII transition. A possible modification to the present protocol could be the addition of 3-isobutyl-1-methylxanthine (IBMX) to the M2 medium used for COCs isolation. IBMX prevents the imme.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Folligon	Intervet	A201A02	Hormonal treatment
Hoechst 33342	Sigma-Aldrich	B2261	For oocyte heterochromatin staining
Cell culture Petri-dish 35 mm x 10 mm	Corning	430165	For COCs isolation
EmbryoMax M2 Medium (1X), Liquid, with phenol red	Merck-Millipore	MR-015-D	For COCs isolation
MEM Alpha medium (1X) + Glutamax	Sigma-Aldrich	M4526	For oocyte in vitro maturation
Cell culture Petri-dish 35 mm glass-bottom	WillCo	GWSt-3522	For imaging experiments
BioStation IM-LM	Nikon	MFA91001	Live cell screening system
Pasteur pipette	Delchimica Scientific Glassware	6709230	For follicles manipulation
Mineral oil	Sigma-Aldrich	M8410	To prevent contamination and medium evaporation
Penicillin / Streptomycin	Life Technologies	15070063	To prevent medium contamination
Fetal Bovine Serum (FBS)	Sigma-Aldrich	ML16141079	For making up αMEM medium
L-Glutamine	Life Technologies	25030	For making up αMEM medium
Taurine	Sigma-Aldrich	T0625	For making up αMEM medium
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3310	For making up αMEM media
Sodium pyruvate	Sigma-Aldrich	P4562	For making up αMEM media
Zoletil (Tiletamina and Zolazepan cloridrate)	Virbac Srl	QN01AX9	For mice anesthesia
Cell_PIV sofware	Kindly provided by Dr. Shane Windsor, University of Bristol, UK	-	-
MATLAB	The MathWorks, Natick, MA	-	For multi-paradigm numerical computing