Summary of patient information.

<p>Summary of patient information.</p

PGCLCs derived from hc-iPSCs.

<p>Immunofluorescent staining of (A) BLIMP1 (green color) and (B) OCT4 (green color) in 4-day-old EBs cultured with PGC induction medium on hc-iPSC-1, -2, -5 and -6 (third to fifth column), PBMC derived iPSC0102 and iPSC0207 (right two column). H9 hESCs (left column) adapted in 4i medium was also detected for BLIMP1 and OCT4 antibodies. Noted that there was no BLIMP1 signals detected in undifferentiated H9 in 4i condition. All samples were doubly stained with SOX17 antibody (red color) sequentially. DAPI was used for counterstain (blue). Scale bar = 50μm. (C) Quantification of immunofluoresent staining of PGCLCs. BLIMP1/SOX17 and OCT4/SOX17 double positive cells were counted for PGCLCs and normalized with the number of DAPI. Percentage of PGCLCs per EB of all lines was presented and significance was compared with control hESC H9. ** indicates P<0.005. *** indicates P<0.001.</p

<i>In vitro</i> characterizations of hc-iPSCs.

<p>(A) The growth curve of hc-iPSCs (hc-iPSC-1, 2, 5, 6), and hESC H9 (3 replicates per line). (B) Population doubling time of hc-iPSC lines and hESC H9. (C) Alkaline phosphatase activity was detected in hc-iPSC-1, -2, -5 and -6 (Blue color, scale bar = 1 mm). Immunofluorescent with antibodies against OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81 were also detected in hc-iPSC lines. Scale bar = 100μm. (D) Flow cytometry confirmed the percentage of SSEA4 and TRA-1-60 positive cells in hc-iPSC lines (red color), as the numbers on graphs indicated, respectively. Isotype controls were shown in white color. (E) Semiquantitative PCR for expression of pluripotent genes. Exogeneous <i>NANOG</i> and <i>POU5F1</i> (<i>Exo-NANOG</i>, <i>Exo-POU5F1</i>) genes were undetectable and endogeneous genes (<i>Endo-NANOG</i>, <i>Endo-POU5F1</i>) were expressed in hc-iPSC lines and hESC H9. cDNA of cumulus cells (CCs) at passage 3 was from mixture of patients. (+): template plasmids which containing <i>POU5F1</i> or <i>NANOG</i>. An unexpected band was amplified by endogeneous NANOG primer in <i>NANOG</i> plasmid with incorrect size of PCR product, which may due to sequence similarity. (-): negative control without template. <i>GAPDH</i> was used for internal control. (F) X chromosome inactivating transcripts <i>XIST</i> were detected in mixture of CCs at passage 0 (P0 CCs), and passage 3 (P3 CCs), but nearly undetectable in hc-iPSCs. (G) hc-iPSCs showed normal female karyotypes without chromosome translocation, deletion or replication.</p

Derivation of hc-iPSCs.

<p>(A) Human cumulus-oocyte-complex (COC). (B) COCs treated with hyaluronidase for 1 minute. (C) Denudated oocyte. (D) Completely dissociated CCs. (E) Morphology of attached human CCs under phase contrast microscope. (F) Cultured CCs of spindle-like shape after passage. (G) Primarily formed iPSC colony after 25 days of induction. (H) hc-iPSCs after manually pick-up. Scale bar: 100 μm.</p

<i>In vitro</i> differentiation and teratoma assay.

<p>(A) The germlayer specific markers SOX17 (endoderm, red color), TUJ1 (ectoderm, green color), and the BRYCHURY (mesoderm, red color) were detected after <i>in vitro</i> differentiation in hc-iPSCs. DNA was stained by DAPI (blue). (B) Expression of germlayer specific genes (<i>SOX17</i> for endoderm, <i>PAX6</i> for ectoderm, and <i>HAND1</i> for mesoderm) were all upregulated in day 10 EBs compared with hc-iPSCs. (C) Ciliated epithelium (endoderm), neuronal-like (ectoderm) and cartilage (mesoderm) cells were present in hc-iPSC derived teratomas (stained with H&E). Arrows indicate corresponding cell types.</p

Collection of metaphase oocytes at different time points.

<p>(A) MII COCs were harvested 15 hours after hCG trigger from oviducts. (B) The cumulus cells were removed from MII COCs. (C) MI COCs were harvested 6–7 hours after hCG trigger from ovaries. (D) The cumulus cells were removed from MI COCs. Scale bar = 50 μm.</p

Schematic illustration of treatments in FRESH-MII (Control 1), VW-MII (Control 2), V/W-AFTER-IVM, and V/W-BEFORE-IVM groups.

<p>The duration of oocyte maturation was 15 hours in all groups, which used hCG trigger as a set point. In FRESH-MII group, the freshly collected IVO MII oocytes were injected with a spermatozoa without any vitrification. In VW-MII Group, IVO MII oocytes were harvested 15 h after the hCG trigger from the oviducts, and those IVO MII oocytes were vitrified for later ICSI. In V/W-AFTER-IVM Group, cumulus cells were removed from MI oocytes, which collected 6–7 h after the hCG trigger from ovaries. After another 8 h of <i>in vitro</i> maturation, the oocytes with a PB extruded (IVM MI-II oocytes) were vitrified for later ICSI. In V/W-BEFORE-IVM Group, cumulus cells removed MI oocytes, which collected 6–7 h after the hCG trigger from ovaries, were vitrified for storage. After oocyte warming, the MI oocytes were <i>in vitro</i> matured for another 8 h, and the oocytes with a PB extruded (IVM MI-II) were regarded as mature oocytes for ICSI.</p

<i>In vitro</i> development of vitrified oocytes after ICSI.

<p><i>In vitro</i> development of vitrified oocytes after ICSI.</p

Preimplantation embryo development of FRESH-MII, VW-MII, V/W-AFTER-IVM and V/W-BEFORE-IVM oocytes following ICSI.

<p>(A) FRESH-MII oocytes. (B) MII-V/W oocytes after warming. (C) V/W-AFTER-IVM oocytes. (D) V/W-BEFORE-IVM oocytes. (E) Cleavage stage embryos derived from FRESH-MII oocytes. (F) Cleavage stage embryos derived from VW-MII oocytes. (G) Cleavage stage embryos derived from V/W-AFTER-IVM oocytes. (H) Cleavage stage embryos derived from V/W-BEFORE-IVM oocytes. (I) Blastocyst stage embryos derived from Fresh-MII oocytes. (J) Blastocyst stage embryos derived from VW-MII oocytes. (K) Blastocyst stage embryos derived from V/W-AFTER-IVM oocytes. (L) Blastocyst stage embryos derived from V/W-BEFORE-IVM oocytes. Scale bar = 100 μm.</p

<i>In vivo</i> development of vitrified oocytes after ICSI.

<p><i>In vivo</i> development of vitrified oocytes after ICSI.</p