An efficiency comparison of different in vitro fertilization methods: IVF, ICSI, and PICSI for embryo development to the blastocyst stage from vitrified porcine immature oocytes

Abstract

BackgroundMost studies carried out to evaluate recovery and development after porcine oocyte vitrification, reported better rates when cryopreserved in embryonic development stages or zygotes, but not in immature oocytes. For this reason, many studies are performed to improve immature oocyte vitrification protocols testing the use of different cryoprotectant concentrations, cooling devices, incubation times; but only a few of them have evaluated which fertilization procedure enhances blastocyst rates in vitrified oocytes. Therefore, this study was aimed to evaluate: 1) if the sperm selection with hyaluronic acid (HA) or polyvinylpyrrolidone (PVP) before injection could play a key role in increasing fertilization and blastocyst formation and 2) the embryo developmental ability and blastocyst production of porcine immature oocytes retrieved after vitrification-warming and co-cultured with granulosa cells during IVM, using different fertilization techniques: in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and conventional ICSI with hyaluronic acid (HA) sperm selection, known as physiological intracytoplasmic sperm injection (PICSI) and.ResultsSperm selected with HA-PICSI displayed a higher percentage of live/acrosome reacted status compared to those in control and exposed to PVP. Higher dead/acrosome reacted rates were obtained after PVP exposure compared to control and HA. In oocytes, viability significantly decreased after IVM in vitrified oocytes. Besides, IVM rates were not different between control denuded oocytes cultured with granulosa cells (DO-GC) and vitrified oocytes. Regarding fertilization parameters, IVF showed higher percentages of total fertilization rate than those obtained by ICSI and PICSI. However, results demonstrate that PICSI fertilization increased the blastocysts formation rate in control DO-GC and vitrified oocytes compared to IVF and ICSI.ConclusionsTo achieve high blastocyst formation rates from vitrified GV oocytes, it is recommended that sperm should be selected with HA instead of PVP before injection since high viability and acrosome reaction rates were obtained. Also, PICSI fertilization was the best method to produce higher blastocyst rates compared to the IVF and ICSI procedures.

Acrosome reaction and chromatin integrity as additional parameters of semen analysis to predict fertilization and blastocyst rates

“…However, in cases of unexplained infertility or poor semen quality, low oocyte retrieval, or previous failed IVF cycles, highly complex techniques as the injection of one spermatozoon into the oocyte cytoplasm (ICSI) are performed [ 3 ]. Recently, new methods are available to improve the quality of the selected spermatozoon, such as hyaluronic acid (HA)-binding sperm selection (PICSI), magnetic-activated cell sorting (MACS) of non-apoptotic spermatozoa, and motile sperm organelle morphology examination (MSOME) techniques [ 4 – 6 ]. PICSI has shown some advantages over IVF and ICSI, such as higher percentage of viable and acrosome reacted cells and higher blastocyst rate in a porcine model [ 4 ]; and in humans, higher fertilization, blastocyst cleavage, and clinical pregnancy rates have been reported using the PICSI method [ 7 ]; however, some of these techniques increase the time of the fertilization procedure, have high costs or fail to determine genetic quality.…”

Effect of porcine immature oocyte vitrification on oocyte-cumulus cell gap junctional intercellular communication.

“…COCs were washed three times in 500 µL drops of maturation medium composed of TCM-199 with Earle´s salts and 26.2 mM sodium bicarbonate (In Vitro, Mexico City) and supplemented with 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine and 10 ng/mL EGF. For IVM, groups from 30 to 50 COCs were placed in each well of a four-well dish (Thermo-Scienti c Nunc, Rochester NY) containing 500 µL of maturation medium supplemented with 0.5 µg/mL LH and 0.5 µg/mL FSH for 44 h [3]. Oocyte culture was performed under mineral oil and incubated at 38.5 °C in an atmosphere of 5% CO 2 in air and humidity at saturation.…”

“…Then, groups of seven oocytes were placed in a 2 µL drop of the second vitri cation solution and loaded into the Cryolock device (Importadora Mexicana de Materiales para Reproduccion Asistida S. A. de C.V., Mexico) in less than 1 min. Then, the Cryolock device was immediately plunged horizontally into liquid nitrogen and stored during 30 min [3]. Warming was performed by the one-step method [10].…”

“…Premature meiotic progression [1], low maturation [4,5], and embryo development rates [3,14] have been reported after the vitri cation of porcine germinal vesicle (GV) immature oocytes. So far, only one study has reported the birth of live offspring from vitri ed GV oocytes in pigs [15].…”

Proteomic Changes of Porcine Oocytes After Vitrification and Subsequent in vitro Maturation: A Tandem Mass Tag-Based Quantitative Analysis

“…With the continuous improvement of their cryosurvival rate (Wu et al, 2017;Appeltant et al, 2018), porcine GV oocytes seem to be more suitable for vitrification. However, the blastocyst yield of vitrified oocytes is still very low as compared with fresh oocytes, regardless of whatever strategy for in vitro embryo production is chosen (Fujihira et al, 2004;Nohalez et al, 2015;Wu et al, 2017;Casillas et al, 2018). This implies that vitrification may produce a certain degree of sublethal damages in the oocytes, thus hindering subsequent embryo development.…”