Identification and in vitro characterization of follicle stimulating hormone (FSH) receptor variants associated with abnormal ovarian response to FSH

Abstract

Context: FSH mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation in women undergoing infertility treatment. The ovarian response of women to FSH is variable, ranging from poor response to ovarian hyperstimulation.

Objective: We investigated whether genetic alterations of the FSH receptor (FSHR) contribute to this variability.

Design and patients: Our approach was to study women undergoing treatment with in vitro fertilization falling into the edges of the normal distribution of ovarian response to FSH, with respect to age.

Setting: We conducted the study at the Yale Fertility Clinic.

Methods: We extracted RNA from cumulus cells surrounding the oocytes of women undergoing in vitro fertilization and analyzed the FSHR mRNA by RT-PCR and sequencing.

Results: We identified four abnormal FSHR splicing products (three exon deletions and one intron insertion) in the FSHR mRNA in 37% (13 of 35) of women tested. All alterations affected the extracellular ligand-binding portion of the receptor without causing a frameshift. When transfected in HEK293T cells, all four splicing variants showed markedly decreased cAMP activation compared to controls. Untransfected cells showed no response to FSH, whereas all the cell lines showed normal cAMP activation when treated with forskolin, a nonreceptor-mediated cAMP stimulant. None of the normal or mutant forms showed any response to LH or TSH.

Conclusions: Our findings strongly indicate FSHR variants as being an intrinsic genetic cause of some forms of infertility and identify a need for functional characterization of these variants and the investigation of more individualized ovarian stimulation protocols.

Figure 1

Oocytes retrieved per cycle after FSH stimulation. A, Cumulative results from the Yale Fertility Center (2003–2005). The number of oocytes that mark the 25% and 75% interval of each age category is underlined. These numbers define low and high responses to FSH in our patient population. B, A graph depicting the data from patients under 35 yr of age. The gray areas represent the lower and higher 25th percentiles of the number of oocytes per cycle.

Figure 2

RT-PCR analysis of FSHR. A, Schematic representation of RT-PCR primers used in nested PCR to amplify the FSHR cDNA. The numbered boxes indicate FSHR exons. B, RT-PCR products from patients carrying FSHR splicing variants. Upper and lower panels show amplification from exons 1–4 and 4–10, respectively. C, Schematic representation of the splicing variants detected in patients. D, Schematic representation of FSHR protein. The NH₂ terminus constitutes the hormone binding domain, is extracellular, and is encoded by exons 1–9. The remaining protein is encoded by exon 10. The COOH terminus is intracellular. The positions of the two common polymorphisms are indicated by black circles. c, cDNA; p, protein. The nomenclature is according to the Human Genome Organization (HUGO) guidelines. E, Distribution of the splice variants along the graph of Fig. 1. del, Deletion; ins, insertion; ivs, intervening sequence (intron).

Figure 3

FSHR mRNA sequencing. The bands shown in Fig. 2B were gel purified and sequenced. The lower panel of each pair shows the sequence of the normal band, and the upper panel shows the variant band. The limits of exons are indicated with a vertical double arrow. A schematic representation of each product is shown above and below each sequence pair.

Figure 4

Haplotype analysis of the variant band. An RT-PCR from exons 4–10 (primers 6F-10R) including the common c.919A/G polymorphism on exon 10 was performed from patients FR-1 and FR-64 who are heterozygous for this SNP. The normal and variant bands were isolated and sequenced. The location of the c.919A/G SNP is indicated with a star above the sequence. The top panel shows the sequence of exon 10 of the variant band. The middle panel shows the normal band. The bottom panel shows a genomic PCR of exon 10 to demonstrate that the patient was heterozygous. The variant band originated exclusively from one of the two alleles. The normal band originated from both. The graph depicts the preferential production of the variant band from one of the two FSHR alleles.

Figure 5

A, cAMP increase upon FSH stimulation. HEK293T cells were transiently transfected with expression plasmids carrying FSHR variants. cAMP was measured after FSH and LH stimulation. Only the two WT constructs (TN, AS) showed cAMP increase. Forskolin activation was used as a positive control. None of the constructs show stimulation with LH or TSH. B, Western blot analysis of transient transfections with the WT and variants of FSHR. The receptor was tagged with HA in the carboxyl terminus, and the detection was done with an anti-HA antibody.