G ivf

On the day of oocyte retrieval, sperm analysis was performed according to the WHO laboratory standards of human semen and sperm. Motile spermatozoa were selected using a Sil Select two layer gradient centrifugation (FertiPro, Beernem, Belgium) for 15 min followed by two 5 min cleansings in G-IVF (Vitrolife, Goteborg, Sweden).
Our sperm criteria required in order to perform the 4 first IUIs were the obtaining of at least 1 million motile spermatozoa, at least 4% of normal sperm and ≥32% progressive motility based on strict sperm morphology criteria (WHO), in the final sperm preparation aliquot (500 μl).
Our criteria for the final sperm preparation to perform classical IVF fertilization are 1–1, 5.10⁵ motile spermatozoa per millilitre and >4% morphologically normal spermatozoa. If the final preparation does not meet these criteria during the first IVF cycle, ICSI is performed and this couple is excluded from the study.

Ejaculated samples were obtained after 3–5 days of abstinence. Semen was screened by the direct upstream method of fertilization culture fluid (G-IVF, Vitrolife Sweden AB). The pellet was resuspended in 0.3 mL fertilization culture fluid.

Sperm was collected from 30 C57Bl6 × CBA (CBAF1) male mice (8–10 weeks of age) from vas deferens and the caudal epididymis. Sperm were collected in 1 ml of pre-equilibrated medium (G-IVF, Vitrolife, Goteborg, Sweden) at 37°C and allowed to swim out for 10 mins. After 10 mins sperm from individual males were split (500 µl each) into either control G-IVF medium or G-IVF medium supplemented with 1500 µM H₂O₂ (this concentration increases ROS levels to a similar level as that seen in an obese male mouse, data not shown) for 1 h, such that both treatments used sperm from the same individual animal. After 1 h of incubation sperm samples were assessed for motility, intracellular ROS levels, sperm binding and then used for in vitro fertilisation.

Embryos were cultured according to conventional methods. After the oocytes were collected, the cumulus-oocyte complex was cultured in fertilization medium (G-IVF; Vitrolife). A discontinuous gradient solution (45% and 90%; SpermGrad, Vitrolife) was used to wash the spermatozoa, and the obtained sperm pellet was placed at the bottom of the fertilization medium. In the IVF group, the optimized upper sperm suspension and cumulus-oocyte mixture were combined and cultured in the fertilization medium for short-term fertilization. After 3 hours, the granular cells were removed by mechanical action using an in vitro fertilization micromanipulation tube (Cook Vandergrift Inc.). Embryo culture was performed using an integrated embryo culture time lapse microscopy system (Embryo Scope; Vitrolife), where images were taken every ten minutes in seven different focal planes. In the ICSI group, the cumulus-oocyte complex was placed in contact with human recombinant hyaluronidase (80 IU/mL) for a short time, and the granulosa cells were peeled off by mechanical action. After ICSI, oocytes were transferred to a prepared TL dish and cultured in the time lapse microscopy system. All the embryos were cultured in a culture device under the same conditions.

The COH protocols included the antagonist protocol, long agonist protocol, mild stimulation protocol, progestin-primed ovarian stimulation (PPOS), and ultra-long agonist protocol [17 (link)]. Recombinant human chorionic gonadotropin (r-hCG) (Merck Serono, Germany), triptorelin (Ferring, Germany), or a combination of both r-hCG and triptorelin was used for triggering to promote oocyte maturation when the diameters of at least two dominant follicles reached 18 mm. Oocytes were retrieved 36 to 38 h thereafter. IVF or ICSI was performed 39 to 40 h after ovulation induction, and the pronuclear (PN) check for fertilization was performed 16 to 18 h later. The culture medium used in the study were G-IVF (Vitrolife Sweden AB, Sweden), G-1 (Vitrolife Sweden AB, Sweden), and G-2 (Vitrolife Sweden AB, Sweden). The incubator conditions were 6% CO₂, 5% O₂, and 89% N₂. The oocytes were placed in G-IVF for fertilization and transferred to G-1 after 16 to 18 h later. Every embryo was cultured to day 3 and scored according to ASEBIR consensus [18 (link)] by two embryologists. Grade A and grade B embryos were good-quality embryos: grade A: 7–8 symmetrical blastomeres with fragmentation ≤ 10%, no multinucleation; grade B: 7–8 symmetrical blastomeres with fragmentation > 11% and ≤ 25%, or ≥ 9 cells with symmetry and ≤ 25% fragmentation. The rest were LGCEs.

The retrieved cumulus-oocyte complexes (COCs) were incubated in pre-equilibrated fertilization medium (G-IVF; Vitrolife, Sweden) at 37°C under a controlled atmosphere of 6% CO₂ and 5% O₂ in an incubator for 2 h until oocyte denudation. The time between HCG injection and oocyte denudation was approximately 40 h. The COCs were transferred into a medium with 80 IU/mL hyaluronidase (Sigma, United States) to disperse the cumulus cells, which were then carefully removed using a denuding Pasteur pipette with decreasing inner diameters (170–140 μm). The morphology of each oocyte was evaluated under an inverted microscope (Eclipse TE 300; Nikon) before ICSI. At high magnifications, SERCs in the cytoplasm appeared round, flat, and clear (Figure 2). Sperm injection into the aggregate was avoided during the ICSI procedure.

Oocytes were obtained from the ovaries or oviducts of 7–10-week-old C57BL/6 J female mice. GV oocytes were harvested from the minced ovaries 48 h after the mice were administered 10 IU of pregnant mares’ serum gonadotrophin (PMSG, Solarbio, P9970) intraperitoneally. The GV oocytes were cultured in G-IVF (Vitrolife) for 4–6 h, and then MI oocytes were collected. To obtain MII oocytes, mice were administered 10 IU human chorionic gonadotrophin (hCG, Univ, 230734) intraperitoneally, 44–48 h after injection of 10 IU of PMSG (Solarbio, P9970). Twelve h later, the MII oocytes were picked under a microscope by fallopian tube dissection. One-cell embryos were collected from 7 to 8-week-old C57BL/6 J females mated with C57BL/6 J males, and ovulation was induced as described above. The embryos were flushed from the reproductive tract 24 h after hCG administration. For all samples collected, the zona pellucida (ZP) was removed by treatment of 10 IU/ml Hyaluronidase (Sigma, H6254).