Fig 2: Abnormal estrogen secretion and atypical endometrial hyperplasia caused by Gdf9 variants. A Serum estradiol levels in Gdf9wt/wt(n=3), Gdf9S415T/S415T (n=3) and Gdf9Q308X/S415T (n=3) mice at 10-12 weeks old during proestrus. *P < 0.05. B Serum testosterone levels in Gdf9wt/wt(n=3), Gdf9S415T/S415T (n=3) and Gdf9Q308X/S415T (n=3) mice at 10-12 weeks old during proestrus. C The mRNA expression of Fshr, Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b2, and Cyp19a1 of ovaries obtained from Gdf9wt/wt (n=3) and Gdf9Q308X/S415T (n=3) mice at 10-12 weeks old. *P < 0.05. D The mRNA expression of Fshr, Lhcgr, Star, Cyp11a1, Hsd3b2, and Cyp19a1 of mouse granulosa cells collected 48 hours after PMSG stimulation. *P < 0.05. E STAR (yellow) and DAPI (blue) immunofluorescence in Gdf9wt/wt and Gdf9Q308X/S415T ovaries. Small (diameter ≤300μm) and large (diameter >300μm) follicles were shown respectively. FD, follicle diameter. F H&E staining of uteri obtained from young (10-12 week) and old (32-34 week) Gdf9wt/wt and Gdf9Q308X/S415Tmice at the stage of proestrus

Fig 3: Altered gene expression in granulosa cells of Gdf9Q308X/S415T mice explored by RNA sequencing. A Differentially expressed genes (DEGs) in granulosa cells collected from Gdf9wt/wt and Gdf9Q308X/S415T mice (three samples in each group, and each samples contains granulosa cells from three mice) 48 hours after PMSG stimulation. DEGs were defined as genes with adjusted P value < 0.05 and |log2FC|>1. B,C Enrichment analysis of GO-BP (Gene Ontology- Biological Process) terms (B) and Reactome pathways (C) using GSEA method. Normalized enrichment score (NES)>0 represent terms/pathways up-regulated in Gdf9Q308X/S415T mice. NES<0 represent terms/pathways down-regulated in Gdf9Q308X/S415T mice. D FPKM (Fragments Per Kilo bases per Million reads) of Has2, P4ha2, Cldn15, and Aqp5 in granulosa cells collected from Gdf9wt/wt and Gdf9Q308X/S415T mice as a result of RNA-sequencing. *P < 0.05. E Expression of P4ha2 in granulosa cells of Gdf9wt/wt (n=3) and Gdf9Q308X/S415T (n=3) mice after 48 hours of PMSG stimulation detected by qPCR. *P < 0.05. F P4HA2 immunostaining in ovaries of Gdf9wt/wt and Gdf9Q308X/S415T mice. The integrated density of P4HA2 staining of cumulus cells (CC) and mural granulosa cells (GC) were calculated separately. *P < 0.001. G mRNA levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours. SB-431542 is an inhibitor of ALK5, which is the receptor of GDF9. *P < 0.001. H Protein levels of P4HA2 in human primary luteinized granulosa cells treated in vitro with GDF9 protein with or without 10μM SB431542 for 24 hours and 48 hours. Meanwhile, protein levels of STAR and CYP19A1 after 48 hours treatment were shown. *P < 0.05

Fig 4: Effects of GDF9 variants on gene expression and GDF9-BMP15 interactions. A Western blots of mature GDF9 proteins expressed in follicular fluid collected from the first and second retrieval cycle of individual P1 and age-matched controls (n=3). B ELISA of GDF9 in follicular fluid collected from the first retrieval cycle of individual P1 and age-matched controls (n=6). C Western blots of GDF9 expression in HEK293T cells transfected with human GDF9WT, GDF9Q321X, GDF9S428T, and GDF9His209GlnfsTer6 respectively and their relative mature-GDF9 expression in culture medium (CM) (3 independent experiments). The amount of concentrated culture medium used for the detection was 35μl per sample per assay. lnfsTer6, His209GlnfsTer6. *P < 0.05. D Western blots of GDF9 precursors and mature GDF9 over time during in vitro cleavage assay (3 independent experiments). Total GDF9=GDF9 precursor+mature GDF9. E Western blots of BMP15 expression in HEK293T cells co-transfected with human BMP15 and human GDF9WT, GDF9Q321X, GDF9S428T, and GDF9His209GlnfsTer6 respectively and their relative BMP15 precursor expression in CM (3 independent experiments). lnfsTer6, His209GlnfsTer6. *P < 0.05. F Co-IP assay of the binding of GDF9 proteins to BMP15 protein. lnfsTer6, His209GlnfsTer6. *P < 0.05. G Western blots of p-smad2 and smad2 expression in human primary luteinized granulosa cells treated with purified recombinant pro-GDF9WT-BMP15 and pro-GDF9S428T-BMP15 protein respectively (3 independent experiments)

Fig 5: Identification of GDF9 bi-allelic variants. A Pedigrees of two affected families. Squares and circles represent males and females respectively. Diamonds represent members whose gender is unknown. Solid symbols indicate the affected members, and open symbols represent unaffected members. The equal sign indicates infertility. Arrows indicate probands. Sanger sequencing chromatograms of GDF9 are shown below. Downward arrows indicate the corresponding variants. B Schematic map of the variant positions in GDF9 at the genomic and protein levels. C Conservation of the affected amino acids is indicated by the alignment of seven species. Red letter represents amino acids affected by the variants. Asterisk indicates that the amino acid is conserved between species