Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles

Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes?

PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring.

In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence.

The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups.

Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique.

Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 μm versus 73.1 μm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl₂). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers.

N/A.

Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells.

PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer.

A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.