Purpose Intracytoplasmic sperm injection (ICSI) is the most widely utilized assisted reproductive technique (ART) worldwide. In this feature, we review the early assisted fertilization attempts that eventually led to the development of ICSI, and discuss its current utilization in cases of male and non-male factor infertility. Methods We researched the literature related to the development, indications, and current use of ICSI, such as sperm structural abnormalities, male genetic indications, surgically retrieved sperm, high sperm chromatin fragmentation, oocyte dysmorphism, and preimplantation genetic testing (PGT). We also describe the potential future applications of ICSI. Results This review summarizes the early micromanipulation techniques that led to the inception of ICSI. We also explore its current indications, including non-male factor infertility, where its use is more controversial. Finally, we consider the benefits of future advancements in reproductive biology that may incorporate ICSI, such as in vitro spermatogenesis, neogametogenesis, and heritable genome editing. Conclusion The versatility, consistency, and reliability of ICSI have made it the most prevalently utilized ART procedure worldwide.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Study question Are haploid genome replication and somatic cell haploidization feasible mechanisms for generating parentally genotyped oocytes? Summary answer Artificial oocytes can be generated by haploid genome replication and somatic cell haploidization. The latter is more efficient and capable of generating live offspring. What is known already A low number of mature oocytes is one of the major limitations to treating infertile women who have impaired ovarian reserve. Although it has been proposed that competent oocytes can be created by a phenomenon known as somatic cell haploidization (SCH), its clinical value has yet to be examined due to its poorly understood mechanism. On the other hand, spindle transfer has been clinically applied for mitochondrial replacement therapy. Therefore, we propose to utilize G2-phase haploid pseudo-blastomere (HpB), generated by parthenogenesis, as a nuclear donor to create oocyte replica. Study design, size, duration In the past 7 months, individual G0 phase cumulus cells (CCs) were transferred into 1,066 ooplasts for SCH. HpBs obtained from the activation of 80 oocytes were transferred into 464 ooplasts. Both cohorts were ICSI-inseminated and placed in the time lapse for embryo development. Another 379 unmanipulated oocytes were ICSI-inseminated, serving as control. Pre-implantation development was monitored and compared for both neogametogenesis techniques. Fully expanded blastocysts were transferred to obtain live pups. Participants/materials, setting, methods CCs were isolated from the cumulus oophorus of B6D2F1 mice. HpBs were obtained via oocyte activation, cultured to the 8-cell stage, and subsequently treated by nocodazole to synchronize at the G2-phase. In two experimental groups, CCs or HpBs were individually transferred into the perivitelline space of the ooplasts with inactivated Sendai virus. Reconstructed oocytes presenting with a pseudo-meiotic spindle were fertilized by piezo-actuated ICSI. Blastocysts were transferred into a pseudo-pregnant CD–1 surrogate to obtain pups. Main results and the role of chance A total of 1,769 oocytes underwent enucleation to generate ooplasts, with a survival rate of 97%. Survived ooplasts were allocated to SCH (n = 1,034) and HpB-SCNT (n = 458). To generate HpBs, 80 unmanipulated oocytes were activated; 58 of them progressed to the 8-cell stage and generated 464 HpB for SCNT. For SCH, CCs were selected based on morphology with a diameter <10 micron. Nuclear transfer of CCs and HpB yielded survival rates of 98.6% and 93.2%, respectively. Following SCH and HpB-SCNT, spindle development for SCH and HpB-SCNT was comparable at 63.5% for SCH and 66.7% for HpB-SCNT. The ICSI survival rates for SCH and HpB-SCNT were 58.9% and 64.9%, respectively, but lower than the control at 73.9% (P < 0.001). Fertilization rates for SCH and HpB-SCNT were also comparable at 61.3% and 64.3%, respectively, but lower than the control at 89.6% (P < 0.00001). Full pre-implantation development was achieved for both experimental groups. While the SCH group yielded a development rate of 24.6% (n = 94), the HpB-SCNT group yielded a lower rate at 12.4% (n = 23) (P < 0.001), both lower than the control (71.7%, P < 0.00001); however, the morphokinetics of the embryo development was retained. To date, only 3 live pups were obtained from SCH group. Limitations, reasons for caution While these techniques to manufacture oocytes are very new and highly experimental, our findings show a lower blastulation rate for oocytes generated by HpB. Both techniques require refinement and improvement of reliability and consistency before they can be considered a feasible technique for human reproduction. Wider implications of the findings: The study confirms the potential to create artificial oocytes capable of supporting full pre-implantation development and, in some cases, live pups. If further streamlining of both procedures demonstrates their safety, they may both represent a viable option to generate de novo gametes Trial registration number N/A

Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P < 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P < 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P < 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P < 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P < 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A