Fertilization medium

Controlled ovarian stimulation in six donors was carried out following an induction protocol consisting in the administration of urinary human follicle-stimulating hormone (Fostipur, Angelini Farmaceutica; Barcelona, Spain), combined with gonadotrophin-releasing hormone antagonist (Cetrotide, Merck-Serono; Madrid, Spain) for down-regulation. The ovarian response was mainly monitored with periodical transvaginal ultrasounds. When at least three follicles with a diameter equal to or greater than 17 mm, 0.4 mg of subcutaneous gonadotrophin-releasing hormone analogue (Decapeptyl, Ipsen Pharma; Barcelona, Spain) was administered as ovulation inducer. Thirty-six hours after GnRH agonist administration, COC (oocyte complexes cumulus-corona-oocyte) were retrieved by transvaginal ultrasound-guided follicular aspiration, and isolated in a pre-warmed buffered medium (G-MOPS, Vitrolife; Goteborg, Sweden). COCs were distributed in four-well dishes (no more than 3–4 oocytes per well) containing 650 μl of Fertilization Medium (CookMedical, Ireland) and incubated at 37 °C in an atmosphere of 6 % CO₂.

Semen samples were collected by masturbation after an abstinence period of 3–5 days and just after egg retrieval by donor ovarian pick-up. After liquefaction, the parameters analyzed included: volume, concentration and motility. The methodology and criteria for assessing semen quality were those established by the World Health Organization (WHO, 2010). Sperm selection was performed in 40 % and 80 % discontinuous density gradients using PURESPERM (Nidacon International AB, Sweden). After 20' centrifugation at 300 x g, the pellet was recovered, washed with 3 ml of Gamete Buffer (CookMedical, Ireland) and centrifuged again for 10 'at 500 x g. Finally, the supernatant was removed and Fertilization Medium (CookMedical, Ireland) was added, adjusting the volume according to on the pellet recovered. The samples were incubated at 37 °C and 6 % CO₂ until insemination [61 (link)].

Five- to nine-week-old female mice were super-ovulated by an intraperitoneal (IP)
injection of 10 IU pregnant mare serum gonadotropin (PMSG; Sigma, St. Louis, MO, USA),
followed 48 hours later by an IP injection of 10 IU human chorionic gonadotropin (Pregnyl,
Organon, Oss, The Netherlands). Sixteen hours after the second injection, the mice were
killed by cervical vertebrae dislocation. The peritoneal cavity was exposed, and the two
oviducts were aseptically removed and placed in Earle’s Balanced Salts Solution (EBSS;
Biological Industries, Kibbutz Beit Haemek, Israel), containing 0.5% bovine serum albumin
(BSA; Sigma, St Louis, MO). Cumulus-oocyte complexes (COCs) were removed from the oviduct
and separated into three groups for the experiment.
To assess the effect of hydrosalpinx fluid on oocytes, two experimental groups and a
control group were studied;100% HSF group: pure hydrosalpinx fluid, 50% HSF group: EBSS
containing 50% of hydrosalpinx fluid, and control group: pure EBSS. The COCs were exposed
to the assigned condition for five minutes. Following the exposure, COCs were washed in
EBSS and transferred to 50 µl drops of fertilization medium (Cook, Brisbane,
Australia) under mineral oil (IrvineScientific, USA) and use for IVF. All steps were done
in an IVF chamber (HD Scientific, NSW, Australia) under an atmosphere of 6% CO₂at 37°C.

Collected COC were washed and incubated individually in Fertilization medium (Cook Medical, USA) under mineral oil for 1 h before denudation. Denudation was performed in 30 μl droplets of Gamete Buffer (Cook Medical, USA) containing 80 IU hyaluronidase (HYASE, Vitrolife, Sweden) for 30 s, and then washed in two 30 μl droplets of enzyme-free Gamete Buffer.
Whenever a BC patient had a male partner, ICSI was performed on a variable proportion of mature oocytes (MII), according to patient’s decision. Embryos obtained were vitrified at 2PN or cleavage stage. If the patient was single, all MII oocytes were directly vitrified after denudation.
In the control group, all MII oocytes were subjected to ICSI. Embryo transfer was performed on day 3 or 5 of culture and the remaining good quality embryos were vitrified according to local protocol. Oocytes and embryos were handled individually in both groups to allow CC per oocyte analysis.