The investigators follow up on the development of children born from ICSI-AOA using the Developmental Red Flags and Ages & Stages Third Edition (ASQ-3) Questionnaires to give strong evidence about the safety of AOA in assisted reproductive technology.

Intracytoplasmic sperm injection (ICSI) is the most widely utilized assisted reproductive technique (ART) worldwide. Fertilization rates obtained after ICSI treatment are reported between 70 and 80%, representing the most efficient ART; however, complete post-ICSI fertilization failure still occurs in 3-5% of cases.

The leading cause of failed fertilization is failure to achieve oocyte activation, a crucial stage in the initiation of embryo development during fertilization. Assisted oocyte activation (AOA) using a calcium ionophore has been used for over a decade following ICSI fertilization failure. AOA is not considered a routine practice of ART yet, which is only suitable for patients with proper indications, including (i) total fertilization failure or low fertilization rate (<30%) in the previous IVF; (ii) severe male factor infertility; (iii) patients with a history of embryo arrest or poor embryo quality in previous IVF cycle.

Regarding technique, the artificial rise of induced calcium rises cannot precisely mimic the physiologically sperm-induced calcium oscillations. Little is known yet about the possible adverse effects of ionophores on post-implantation embryo development. Numerous studies have been conducted to compare the development of children born from ICSI - AOA versus non-AOA. Thus, the investigators performed this study to investigate the physical, mental, and motor development of children born following ICSI - AOA using the Developmental Red Flags and Ages & Stages Third Edition (ASQ-3) Questionnaires.

Inclusion Criteria:

All single, live babies born following ICSI with AOA and ICSI without AOA from 08/2020 to 04/2021.

Parents consent to participate in the study. Group ICSI+AOA: Embryos from ICSI with AOA with calcium ionophore

In the ICSI+AOA group, indications of AOA are:

• Previous failed IVF treatment with no fertilization or poor fertilization rate (fertilized oocytes/pick-up oocytes<35%) or poor embryo result (number of embryos/pick-up oocytes<35%, <3 embryos, no good-quality embryo)
• Using sperm after retrieval technique, cryptozoospermia Group Control: Embryos from ICSI without AOA.

In the Control group, the indications of AOA for the next IVF cycle (if patients continue the next IVF cycle) are:

• Currently failed IVF treatment with no fertilization or poor fertilization rate (fertilized oocytes/pick-up oocytes<35%) or poor embryo result (number of embryos/pick-up oocytes<35%, <3 embryos, no good-quality embryo)

Exclusion Criteria:

Embryos with PGT. Oocyte donation

In the ICSI+AOA group, indications of AOA are:

• Previous failed IVF treatment with no fertilization or poor fertilization rate (fertilized oocytes/pick-up oocytes<35%) or poor embryo result (number of embryos/pick-up oocytes<35%, <3 embryos, no good-quality embryo)
• Using sperm after retrieval technique, cryptozoospermia

In the Control group, the indications of AOA for the next IVF cycle (if patients continue the next IVF cycle) are:

• Currently failed IVF treatment with no fertilization or poor fertilization rate (fertilized oocytes/pick-up oocytes<35%) or poor embryo result (number of embryos/pick-up oocytes<35%, <3 embryos, no good-quality embryo)