In the present work, we demonstrate that human granulosa-luteal cells supplemented with rLH exhibit increased expression of LHR, AR, SOX9, and AMH. This report is the first study to compare the expression profiles of genes in the androgen receptor and anti-Müllerian hormone in pre-ovulatory granulosa cells from IVF/ICSI patients receiving rLH supplementation for COH. Patients with low expression of AR, AMH, and SOX9 may require a different follow-up schedule and corresponding rLH treatment.
Little is known about the extremely complex process that generates a developmentally competent oocyte. During folliculogenesis and oogenesis, the oocyte is surrounded by granulosa cells. The two cell types communicate bidirectionally through secretion of steroid hormones and paracrine factors. This communication plays a key role in folliculogenesis and is essential for an oocyte to achieve fertilization and undergo embryogenesis .
It has been suggested that the expression levels of hormonally-regulated genes in granulosa cells can be used as markers for oocyte quality and hence developmental potential [34, 35]. This development is mirrored by the FSH-induced increase in estradiol biosynthesis in granulosa cells after adequate stimulation of theca cells by LH . In 2004, Westergaard et al. found that the follicular fluid concentrations of LH, estradiol and androstenedione were significantly higher and progesterone levels were significantly lower in women treated with gonadotropin plus rLH relative to women treated with recombinant rFSH alone . Smitz et al. also reported that major differences in the serum and follicular fluid endocrine profiles exist after stimulation with gonadotropin and LH, as exogenous LH activity may induce a different endocrine environment . In agreement with these reports [37, 38], we found that the addition of rLH was associated with increased estradiol, testosterone, and androstenedione and reduced progesterone in the fluid of the dominant follicle.
AMH is detected in serum from women of reproductive age, and the levels vary.
Slightly with the menstrual cycle, reaching a peak value in the late follicular phase. AMH expression follows a similar pattern in humans compared with mice and rats, suggesting an important role for AMH in folliculogenesis . Wunder et al.  reported that serum and follicular fluid AMH levels on the day of oocyte retrieval are correlated with reproductive outcome. From molecular point of view, AMH has been shown to be a downstream target of SOX9. Upregulation of SOX9 can promote the expression of AMH . While increased intrafollicular androgen levels have been associated with significant increases in granulosa cell production of AMH , few studies have investigated how reproductive hormones such as LH influence the interaction between the androgen/AR and AMH/SOX9 pathways in granulosa cells.
We demonstrated that luteinized granulosa cells from IVF patients supplemented with rLH display increased LHR, AR, SOX9, and AMH expression and higher testosterone and androstenedione concentrations in the follicular fluid. Androgens are considered detrimental to the late stages of folliculogenesis , and hyperandrogenic polycystic ovary syndrome (PCOS) is associated with follicular development arrest and poor oocyte quality . It is possible that LH may stimulate ovarian theca cells to produce androgens that act cooperatively with LH in a paracrine fashion on granulosa cells. Our in vitro model showed that DHT alone can downregulate AMH expression, but increased expression of AR and AMH was observed after rLH treatment. Indeed, many factors [44, 45] other than androgens may have affected the expression of AR, AMH and SOX9 under rLH treatment. Increased estradiol or decreased progesterone in the follicular fluid may also influence LH-regulated granulosa cell gene expression. Further studies on interaction between LH and sex steroid hormones will be of interest.
The homogeneity of our study population is an advantage because it reduces the possibility of confounding population substructures or admixtures. However, because our study population was restricted to Taiwanese patients, the results may not be generalizable to other ethnicities. Although our study showed no significant differences in terms of clinical outcomes with or without rLH supplementation (Table 2), the study was underpowered to look at pregnancy rates between the two groups. Our experiments did demonstrate that granulosa-luteal cells supplemented with rLH displayed increased expression of LHR, AR, SOX9, and AMH. As it is difficult to extrapolate the role of rLH in follicular growth and oocyte maturation based on our results, further molecular and cellular functional studies are required to investigate whether rLH therapy has unremarkable benefits or perhaps an undetermined effect on follicular growth and oocyte maturation in women.