A COH protocol with human recombinant FSH (Gonal-F, Merck Serono S.A., Madrid,

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1 Supplemental Methods Detailed protocol for controlled ovarian stimulation A COH protocol with human recombinant FSH (Gonal-F, Merck Serono S.A., Madrid, Spain) was initiated on menstrual cycle day 2 at a fixed dose of 300 IU/day, s.c. Pituitary suppression was achieved by daily s.c.administration of 0.25 mg of cetrorelix acetate (Cetrotide, Merck Serono, S.A., Madrid, Spain) when at least one follicle exceeded a mean diameter of 14 mm, and antagonist was maintained until day of ovulation triggering. When at least 1 follicle reached a mean diameter of 17 mm, ovulation was induced by s.c administration of 0.2 mg of triptorelin acetate (Decapeptyl, IpsenPharma, Barcelona, Spain). Transvaginal oocyte retrieval was performed 36 hours after agonist administration and ICSI was employed in all cases. Embryo quality was recorded according to ASEBIR criteria (Balaban, et al., 2011). One or 2 embryos were transferred on day 2 after collection unless oocyte pick-up took place on Friday or Saturday, in such cases, embryo transfer was performed on day 3. Luteal phase was supported by one s.c.injection of 1500 IU of hcg (Ovitrelle, Merk-Serono, S.A., Madrid, Spain) at oocyte collection and 400 mg/day of vaginal micronized progesterone (Progeffik, Lab. Effik, Madrid, Spain) during the entire luteal phase. If pregnancy occurred, progesterone administration was extended up to the evidence of fetal heart activity at ultrasound. Sonography examinations All ultrasound scans were performed using a Voluson i (General Electric, Madrid, Spain) equipped with a MHz endovaginal 3D probe. Antral follicle count (AFC), included follicles measuring 2-9 mm and it was performed on day 1-4 of the menstrual cycle. Serum and follicular fluid hormonal determinations

2 All blood samples were drawn by venopuncture on the first day of ovarian stimulation and on the day of ovulation triggering. Serum samples were separated by centrifugation at 600g for 10 minutes at room temperature and frozen in aliquots at -20ºC for subsequent centralized analysis. An aliquot of follicular fluid harvested after luteinized granulosa cells (LGCs) isolation by centrifugation process from each patient was stored at -20ºC. Due to higher values of steroids sexual hormones in the follicular fluid, samples had to be diluted with specific diluents at 1/5000 for E2 and P4, 1/25 for T and 1/10 for A4. Platforms, minimal detection limits and inter and intra-assay coefficients of variation are showed in the table below: Plataform Metabolite Detection limit CV intra-assay CV inter-assay Immulite 2000 DHEA-S 0.1 μmol/l 7.6% 8.1% Immunoassay SHBG 0.2 nmol/l 6.5% 8.7% system (Siemens Healthcare Diagnostics, Madrid, Spain) GenII AMH Assay (Beckman Coulter, CA, AMH 0.08 ng/ml <6% <10% USA) Architect analyzer (Abbott Diagnostics, Madrid, Spain) FSH 0.05 IU/L 3% 4% LH 0.07 IU/L 2% 3% E2 10 pg/ml 4.6% 5% T 0.08 ng/ml 4% 5.1% Δ nmol 6% 9% P4 0.1 ng/ml 2% 5% Isolating of putative luteinized granulosa cells from human follicular aspirates Isolation of putative LGCs was performed from individual patient follicular aspirate pools according to the protocol published by Kossowska (Kossowska-Tomaszczuk, et al., 2009). Human follicular fluid (FF) samples were collected from each patient after removal of cumulus oophorus-oocyte complexes (COC) after oocyte retrieval. FFs pooled without obvious blood contamination were processed by centrifugation through a one-step density Histopaque 1077 gradient (Sigma-Aldrich, St. Louis, MO, USA) at 700 g for 30 min at room temperature (RT). Mononuclear cells at the interface layer were removed in order to isolate the putative luteinized granulosa cells (LGCs) and were fixed in 4% paraformaldehyde (BDH Prolabo, UK) for 30 min

3 at RT. Fixed cells were washed by centrifugation at 600g for 10 min at RT and cellular pellet was mechanically dispersed to reduce clumping and resuspended in 1 ml of phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA). Cell number and viability of the luteal cell suspension was tested prior to fixation step using exclusion test with vital dye Trypan Blue staining (Sigma- Aldrich, St. Louis, MO, USA). Quantification of FSHR and AR on the LGC cells by flow cytometry technique Putative LGCs resuspended in PBS-BSA 3% were incubated separately with unconjugated primary antibodies as polyclonal rabbit anti human FSHR (H-190) at a dilution of 1:10 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and monoclonal rabbit anti human AR at 1:60 dilution (Abcam, Cambridge, UK) for 30 min at RT. Following incubation time, the cell suspensions were washed once by centrifugation at 600g for 5 min. Secondary antibody goat anti rabbit Alexa Fluor 488 IgG (Invitrogen, Carlsbad, CA, USA) and a common panleukocyte marker mouse anti human CD45-PC7 IgG1 (Beckman-Coulter, Brea, CA, USA) were added in both cell suspensions at a dilution of 1:300 and 1:20 respectively. Cell suspensions were incubated for 30 min at RT in the dark, washed once and resuspended with PBS. A third cell suspension without primary antibody was used as isotype control so it was incubated with monoclonal mouse anti human MsIgG1-PC7 antibody (Beckman-Coulter, Brea, CA, USA) at a dilution of 1:20 for 30 min at RT in the dark and processed with identical staining. Finally, 5 µg/ml of propidium iodide (PI) was added (Sigma-Aldrich, St. Louis, MO, USA) to exclude necrotic and platelet cells. Flow cytometry analyses were performed using a GALLIOSTM Flow Cytometer (Beckman-Coulter, Brea, CA, USA) using an argon laser tuned at 488 nm and 22 mw. At least 20,000 events per sample were counted by electronic gating on forward and side scatter light parameters. Debris was discarded by scatter properties and dot plots corresponding to the CD45 negative area and PI positive area were purchased to determine the average of labeled LGCs. Mean fluorescence intensity, defined as average fluorescence value of the corresponding staining referred to the logarithmic scale of fluorescence intensity along the X

4 axis of the histograms, and cell count of gated cells denoted as LGCs FSHR+/CD45-/IP+ and LGCs AR+/CD45-/IP+ were analyzed using the CXP system program (Beckman-Coulter, Brea, CA, USA). FSH, LHR, AR and steroidogenesis-related gene expression Cumulus cells (CCs) were mechanically removed from every single oocyte-cumulus-complex of each patient before hyalurinodase stripping step. Cumulus RNA was extracted using the Quick-RNA TM MicroPrep (Zymo Research, Irvine, CA, USA) according to the manufacturer s protocol. Isolated total RNA was reverse-transcribed into cdna using Advantage RT-for-PCR kit (Clontech, Palo Alto, CA, EEUU). Relative gene expression analysis were carried out by fluorescent quantitative real-time polymerase chain reaction (FQ-RT-PCR) (Applied Biosystems, Carlsbad, CA, USA) with proprietary TaqMan probes (Applied Biosystems, Carlsbad, CA, USA) for FSHR, AR, LHR, StAR, CYP11A1 and CYP19A1 in an ABI 7900HT sequence detection system (Applied Biosystems, Carlsbad, CA, USA). A list of the probes is available below. All PCR reactions were performed in duplicate and each study gene was normalized to endogenous gene 18S. Relative gene expression was calculated by ΔΔCt method (Livak and Schmittgen, 2001). mrna probes for expression analysis Target mrna FSH-Receptor Androgen-Receptor LHCG- Receptor Star CYP11A1 CYP19A1 18s PROBE SEQUENCE REFERENCE Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _s1

5 Balaban, B., Brison, D., Calderon, G., Catt, J., Conaghan, J., Cowan, L., Ebner, T., Gardner, D., Hardarson, T., Lundin, K. et al. (2011) The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human Reproduction, 26, Kossowska-Tomaszczuk, K., De Geyter, C., De Geyter, M., Martin, I., Holzgreve, W., Scherberich, A. and Zhang, H. (2009) The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells, 27, Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, Supplemental Figure 1: Flow cytometry quantification of LGCs positive for FSH-receptor (%LGC FSHR+) and androgen receptor (%LGC FSHR+) in follicular fluid from confirmed low responder patients. Confirmed poor responders after a conventional IVF cycle (phase 1) were allocated to receive one of three priming strategies before a second IVF cycle: transdermal testosterone (T), transdermal estradiol (E) or estradiol combined with oral contraceptive pill (EOCP). The boxplot represent median, p25 and p75 of the distribution, the whiskers represent the minimum and maximum values. Comparisons within patients (priming vs non-priming) were done using the Wilcoxon s rank test for paired samples. Comparisons between priming strategies were done using the Kruskall- Wallis test. No significant differences were evidenced for any comparison at a significant level of p<0.05.

Gladstone Institutes, University of California (UCSF), San Francisco, USA

Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24 Gag Marianne Gesner, Mekhala Maiti, Robert Grant and Marielle Cavrois * Gladstone Institutes, University of