Analysis software and apparatus for screening early embryo

A method, comprising:

• subjecting a patient to transplantation of freeze-thawed blastocysts under hormone replacement therapy (HRT) with estrogen and progesterone, comprising
• in a pretreatment cycle, initiating application of gonadotropin-releasing hormone (GnRH) agonist on day 7 of the hyperthermic phase in the cycle before treatment, and from day 3 of menstruation and thereafter, subjecting the patient to daily stimulation of ovaries with follicle-stimulating hormone, until a diameter of the largest subordinate follicle reached a predetermined value, and triggering ovulation,
• intramuscularly injecting human chorionic gonadotropin (hCG), and transvaginally collecting ova under ultrasonographic guidance after the injection, and measuring follicles by ultrasonic scanning,
• fertilizing the collected ova by an insemination method or an intracytoplasmic sperm injection method and culturing fertilized eggs in a medium 1 to obtain early embryos,
• capturing an image of a fertilized egg being cultured such that images of the fertilized egg are captured with a predetermined interval;
• detecting a male pronucleus and a female pronucleus in the images of the fertilized egg captured;
• measuring a diameter in a horizontal direction and a diameter in a vertical direction of the male pronucleus and the female pronucleus;
• calculating respective areas of the male pronucleus and the female pronucleus according to the following formula:
• area of pronucleus=π×½×diameter in horizontal direction×½×diameter in vertical direction (μm2), by designating the maximum width in the horizontal direction of the pronucleus as the diameter in the horizontal direction, and designating the maximum width in the vertical direction as the diameter in the vertical direction,
• wherein the areas of the female pronucleus and the male pronucleus are measured over time with intervals of every 5 minutes to every 1 hour,
• calculating a first area difference between the respective areas of the female pronucleus and the male pronucleus from an image of the fertilized egg captured at a point of time in a period from 1 hour before to 10 hours before a time of occurrence of male and female pronuclear membrane breakdown;
• calculating a second area difference between the respective areas of the female pronucleus and the male pronucleus from an image of the fertilized egg captured at a point of time in a period from immediately before to 20 minutes before the time of occurrence of the male and female pronuclear membrane breakdown;
• and
• selecting a fertilized egg for transplantation based on calculated values of the first area difference and the second area difference, wherein the selecting comprises selecting the fertilized egg that satisfies that the second area difference is less than 40 μm2,
• culturing at least one of the obtained early embryos obtained from the selected fertilized egg in a medium 2 on a culture plate in an incubator, and obtaining blastocysts, and
• transplanting the blastocysts obtained in the culturing of the selected fertilized egg into the uterine cavity, and determining a subsequent birth status of the transplanted patients.