Genetic Sensitivity to Fertility Drugs (Gonadotropins)
A major effort of my research has been utilizing mouse models to investigate genetic variation in sensitivity to fertility drugs, i.e. gonadotropins. The tremendous inter-individual variability in sensitivity to gonadotropins is a major problem with assisted reproductive therapies (ART) in many mammalian species, including humans. While some women fail to respond to gonadotropins, others hyperstimulate to risk multiple pregnancy and the potentially life-threatening ovarian hyperstimulation syndrome. While there is clearly an age-related decline, several studies show that much of the individual variability in ovarian sensitivity to fertility drugs is actually genetic.
As shown in the adjacent figure, we discovered that strains of mice differ 5 to 6-fold in the number of eggs ovulated in response to fertility drugs, i.e. gonadotropins. Where as A/J strain mice ovulate 8 to 9 eggs in response to gonadotropins, C57BL/6J strain mice ovulate 42 to 54 eggs (Spearow 1988; Spearow et al. 1999a). The variation in Ovulation Rate, gonadotropin Induced (ORI) between strains of mice is remarkably similar to the variation in ovarian response to gonadotropins observed in mammalian species ranging from humans to wildlife and livestock. These major differences in ORI observed between mouse strains suggest that much of the incredible variation in ovarian response to gonadotropins in humans and other mammalian species, is actually genetic.
In terms of genetic markers, a common polymorphism in the Follicle Stimulating Hormone Receptor (FSHR) gene was associated with human ovarian response to gonadotropins in some studies (Sudo 2002, Behre et al 2005) but not in others (d’Alva et al 2005). Ser(680)/Ser(680) genotype at the FSH receptor locus was associated with a decreased estradiol response to gonadotropins, and a higher FSH/hMG dose for ovarian stimulation in normal-ovulating women (Sudo 2002; Behre et al 2005), but not in anovulatory women (Laven 2003). However, in vitro expressed recombinant FSH receptor isoforms did not differ in the affinity of FSH binding or cAMP production and other biological endpoints. Thus, although there may be a minor effect of common FSH receptor polymorphisms on ovarian response to gonadotropins, the data suggest that the loci with largest effects on this trait have yet to be identified.
Due to the need for identifying genetic markers for ovarian sensitivity to gonadotropins, we conducted a genetic linkage analysis in mouse backcrosses. The results showed that the 6-fold differences in ORI segregated as though this trait was controlled by the action of approximately 3 to 4 loci, i.e., genes with largest effects (Spearow et al. 1999a). We then mapped statistically significant genes controlling induced ovulation rate to specific regions of mouse chromosome 6 and 10, and statistically suggestive gene to regions of mouse chromosome 2, 9 and X (Spearow et al. 1999b). These genes controlling significant differences in Ovulation rate Induced are referred to as Ovulation Rate Induced Quantitative Trait Loci, or ORI QTL. We also mapped ORI QTL to these and other chromosomal regions in specialized strains of mice, called Recombinant Inbred Strains. These studies revealed that ORI was controlled by directly acting genes and especially by gene-gene interactions, i.e. epistasis (Spearow, In preparation).
Unfortunately the independent segregation of several genes controlling ORI creates considerable genetic noise, making it all the more difficult to identify each locus. The situation is analogous to being in a room where 4 different radios are all playing different songs - all you hear is a jumble of noise. But if you turn off all of the radios except for one - you know it is Beethoven's 5th symphony. Similarly, genetic noise from the segregation of multiple genes controlling a trait also makes it difficult to identify the individual genes involved. This is especially true in humans and other outbred mammalian species, where direct genetic effects, gene-gene interaction effects and environmental effects influence reproductive traits. But I knew that an animal model differing at a single direct or interacting locus would allow us to map each gene controlling the observed differences in sensitivity to gonadotropins. Since there is a real need for improved genetic markers for sensitivity to gonadotropins, I spent several years developing several reproductive congenic strains of mice, each with a single chromosomal region containing a B6 (high) ORI QTL on the A/J low ORI genetic background.
The adjacent figure shows how one of these strains, the A.B6Chr 2 proximal congenic strain, was developed from a AxB6 F1 cross by repeated backcrossing to A/J strain mice. After 10 generations of backcrossing to A/J strain mice with selection for individuals that have B6 (High induced ovulation) alleles in the shaded region of Chromosome 2, 99.9% of the genes in these mice will have the A/J form of the genes, except in the region flanking the genes controlling ORI on Chromosome 2.
Comparing this A.B6 chromosome 2 congenic strain with that of A/J strain mice allows us to map and characterize the gene in this region of chromosome 2 that controls gonadotropin-induced ovulation. We have produced several reproductive congenic strains, each with a different region of B6 high ORI alleles on the A/J low ovulation induction background. This approach eliminates the genetic noise from the segregation of other genes. i.e. loci, elsewhere in the genome and is uniquely suited for identifying the genes controlling ovarian sensitivity to gonadotropins through positional, candidate gene cloning approaches. This involves comparing the gene sequence, level of expression and physiological function of likely candidate genes in this small chromosomal region to determine if such genes control the observed difference in ovarian sensitivity to gonadotropins.
If funding can be obtained to continue these studies, I will use these and other mouse genetic models to map and identify genetic markers for ovarian response to gonadotropins. Once identified in mice, it will be a straightforward process to characterize and test the corresponding genes in humans and other mammalian species as markers for sensitivity to ovulation induction. Such molecular markers would aid in the optimization of hormonal treatments for achieving the desired level of ovarian stimulation while reducing chances of multiple pregnancy and the potentially life threatening Ovarian Hyperstimulation Syndrome (OHSS). Molecular markers would also aide in the optimization of hormonal treatments in other species including endangered species whose recovery may depend on successful ART.
Even though women are undergoing over 100,000 ovarian stimulation cycles per year in the US, and many more in other countries, physicians have difficulty predicting how a woman will respond to gonadotropins until well into her first ovulation induction protocol. While some women fail to respond, others go off the charts and are at risk for multiple pregnancy, premature delivery and OHSS. Closely monitoring serum estrogen levels by immunoassay and follicular development by ultrasound clearly aids in the management of controlled ovarian stimulation. However, this is expensive and poor responders are usually initially treated with too low of a gonadotropin dose, while hyper-responders are frequently initially treated with too high of a gonadotropin dose. The latter have to be coasted or discontinued - at considerable financial cost to the patient- or face the risks of OHSS. Even though couples are spending about $10,000 or more per ovulation induction/ IVF cycle, current methods can not accurately predict how a women will respond to the fertility drug treatments until well into to the treatment protocol. It is important to realize that genetic markers for ovarian responsiveness to gonadotropins would aid in optimizing ovarian stimulation / ovulation induction treatments, thereby improving the efficacy and safety of ovarian stimulation in humans and other species, including livestock and endangered wildlife.
Funds are urgently needed to continue this research to identify improved genetic markers for ovarian response to gonadotropins. If you would like to make a Tax-deductible contribution to the University of California at Davis to support this research, please contact Dr. Jimmy Spearow at jlspearow@sbcglobal.net or (530) 902-2041. If you email, please include "Reproductive Genetics" in the subject line.
Thank you.
Jimmy Spearow
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