Pregnyl

We followed institutional guidelines for animal care and research protocols, and approved by the Etiska Nämnden on animal use (ethical permit N241/14). Xenopus laevis eggs were obtained by injecting frogs with 700 units of human chorionic gonadotropin (Pregnyl®, Merck Sharp & Dohme). The embryos were fertilized using a sperm suspension and were dejellied with 1% thioglycolic acid at two-cell stage, and cultured in 0.2× Marc’s Modified Ringer’s solution (MMR) at 18-21 °C. Staging was according to Nieuwkoop and Faber [31 ]. Microinjections were performed in 4% Ficoll/0.3× MMR. The maximum injection volume was 20nl per embryo. The embryos were then cultured in 0.2X MMR until either stage 10.25 (for TOPFlash assay) or stage 32 (for the PCP phenotypes). The mMessage mMachine® sp6 Kit (Ambion) was used to synthesize in vitro capped mRNA. Lrrk2 construct used for the experiments was pCS2-5Xmyc-Lrrk2 [32 (link)]. pCS2-super was generated by inserting an oligonucleotide fragment containing a polylinker sequence (EcoRI, PacI, SbfI, XmaI, XhoI, AscI, XbaI) into the EcoRI/XbaI sites of pCS2. To generate pCS2-beta-galactosidase, beta-galactosidase with nuclear localization signal was obtained by RT-PCR from pBSApBpACAGftILn [33 (link)], and subcloned into the PacI/AscI sites of pCS2-super.

Only couples with normal BMI and no history of smoking, excess drinking, or use of recreational drugs were included. All patients included were Caucasian and had comparable durations and indications of infertility. The average AMH of the female partner was 3.2 ± 3 ng/mL, the average FSH was 5.3 ± 4 IU/mL, and the average peak estradiol for ICSI stimulation was 3066.3 ± 1731 pg/mL.
A similar superovulation protocol was used for all couples. Patients were treated with daily gonadotropins (Follistim; Merck, Kenilworth, NJ, USA; Gonal-F; EMD-Serono, Geneva, Switzerland; and/or Menopur; Ferring Pharmaceuticals Inc, Parsippany, NJ, USA). Precocious ovulation was prevented by GnRH antagonist administration (Ganirelix acetate; Merck, Kenilworth, NJ, USA; or Cetrotide; EMD-Serono Inc., Rockland, MA, USA). The trigger for final oocyte maturation with human chorionic gonadotropin (Pregnyl, Merck) was administered when the two leading follicles reached a diameter of ≥ 17 mm [22 , 23 (link)]. Transvaginal oocyte retrieval was performed under conscious sedation 35–37 h after hCG administration.
Cumulus-corona cells were removed by exposure to medium containing 40 IU/mL hyaluronidase (Cumulase; Halozyme Therapeutics, Inc., San Diego, CA) [24 ] and incubated 1–2 h prior to ICSI.

Ovarian stimulation was carried out using a long luteal gonadotropin-releasing hormone (GnRH) agonist protocol or an antagonist protocol, as described previously[18 (link)], Briefly, once two-thirds of the follicles reached 18 mm, 5,000–10,000 IU of human chorionic gonadotropin (hCG; Pregnyl, Merck) was injected. At 35–36 h after hCG administration, oocytes were collected in G-IVF media (Vitrolife) under transvaginal ultrasound guidance.

All women in this study were stimulated with the short gonadotrophin-releasing hormone (GnRH) antagonist protocol. Ovarian hyperstimulation was initiated using recombinant gonadotrophin, follitrophin beta (PuregonⓇ, Organon Malaysia Sdn Bhd) and was dictated according to patients' responses to stimulation, which was evaluated by serial vaginal ultrasound examinations (Acuson 50, Siemen Healthineers, Erlangen, Germany). When the majority of the follicles reached 13-14 mm in diameter, GnRH antagonist, ganirelix (OrgalutronⓇ, Vetter Pharma-Pertigung GmbH & Co. KG) was added at a dose of 250 μg daily. Once at least 3 leading follicles reached a size of 18 mm, 10,000 IU intramuscular human chorionic gonadotrophin (hCG; PregnylⓇ, Merck Sharp & Dohme, Malaysia) was administered. During this time, the number of mature follicles, endometrial thickness, and level of serum progesterone were determined. Oocyte retrieval was then performed 34-36 hr after the hCG administration. Using the total oocyte scoring system, the oocytes were scored as -1 (poor quality), 0 (almost normal), and +1 (normal) oocytes (17). The semen was prepared using the density gradient method (18), and the most motile and normal sperm was selected for the ICSI procedure.

Patients undergoing IVF/ET treatment initially received controlled ovarian hyperstimulation using medication that prevents the premature luteinization, including GnRH antagonists (ganirelix, Merck, Canada) in the follicular phase or triptorelin acetate (Synarel, Pfizer, Canada) in the luteal phase of the previous cycle to downregulate the pituitary gonadotropin-releasing hormone receptors and subsequent transduction pathways. On the second day of the menstrual cycle, appropriate human menopausal gonadotropin (hMG, Menopur, Ferring, Canada) or recombinant FSH (Puregon, Merck, Canada) were administered to stimulate follicular growth. When the diameter of the leading follicle reached > 18 mm or the diameters of at least three follicles reached > 17 mm, hCG (Pregnyl, Merck) was administered to trigger final oocyte maturation. Oocytes were retrieved under vaginal ultrasound-guiding 34–36 h after the trigger, and corresponding follicular fluid was collected.