Recombinant Luteinizing Hormone. Is it necessary?
It has been known for over 50 years that both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are required to stimulate follicular development and estradiol synthesis. However, previous studies using FSH and LH preparations have not been able to answer unequivocally whether an observed response was solely due to either FSH or LH because they were not pure preparations.
In view of the recent availability of both 100% pure recombinant human FSH and recombinant human LH we now have an unique opportunity to test their contribution on the regulation of the ovarian function.
For some years there has been a debate among scientists and clinicians as to the roles and relative importance of LH and FSH in follicular development. We knew that some level of ovarian LH stimulation is required for the support of steroid biosynthesis that occurs during the gonadotrophin dependent phase of follicular growth.
Circulating levels of LH are essential for the production of steroid hormones to regulate the timing of ovulation and target tissue responses as well as for the maintenance of the corpus luteum and therefore early pregnancy. Elevated levels of serum LH during the follicular phase of the menstrual cycle are not only unnecessary for follicular maturation but are deleterious to normal reproductive process. These elevations may occur as a result of administration of exogenous LH or through an endogenous pathological process (i.e. polycystic ovarian disease, PCOD). We also knew that elevated levels of LH during the preovulatory period may also negatively influence post ovulatory events such as conception and implantation.
In our opinion the best results for ovulation induction can be expected by using purified follicle stimulating hormone (RecFSH) administered after gonadotrophin releasing hormone down-regulation.
The question in this review arises what practical benefit the availability of pure LH might provide.
At the beginning of each ovulatory cycle, the FSH signal from the anterior pituitary gland stimulates a small amount of ovarian follicles to grow. These follicles are endowed with receptors for FSH and are not responsive to LH at this time as they do not posses LH receptors (21). These follicles respond to the initial stimulation of FSH by cell growth and proliferation. FSH stimulates the transcription of genes that encode growth factors. These factors act in a paracrine and autocrine way within the follicular microenvironment. They play an important role in the final maturation of the follicle and its contents. During the mid-follicular phase, following FSH stimulation, the granulosa cells begin to synthesize LH receptors and acquire the ability to respond to the stimulatory effects of LH (20).
LH stimulation of specific cells within the ovary is required for the production of steroid hormones. As described by the two cell theory, LH is critical for the production of androgens from the interstitial theca cells. Androgens produced by theca cells travel to the granulosa cells to be converted to estradiol. During the early follicular phase, LH receptors are found only on ovarian theca cells (22). Following LH stimulation, the theca cells express a series of enzymes that convert cholesterol to androgens. Androgens are converted to estrogens within the FSH-stimulated granulosa cells (6).
Elevation in serum LH levels during the follicular phase of the menstrual cycle have been associated with lower fertility rates.
Pharmaceutical agents that are used to induce the development of multiple follicles may disturb the complex feedback mechanisms that exist within the hypothalamo-pituitary-ovarian axis. Elevated plasma levels of estradiol, produced by the growth of multiple follicles induced by HMG injections, can trigger an untimely endogenous LH surge. This may occur before the individual follicles are fully matured. Thus, a reduction in the number of fertile ova may result from inappropriate steroid production induced by the administration of endogenous gonadotrophins.
Induction of the growth of multiple follicles may also result in an attenuation of the endogenous midcycle LH surge (23). This may result in oocyte maturation without follicular rupture.
Untimely surges of LH at the midcycle or elevated levels of LH during the follicular phase of the menstrual cycle as a result of administration of the gonadotrophins may be deleterious to folliculogenesis, conception or implantation (20).
Homburg et al. reported that in patients with polycystic ovarian disease (POCD), who miscarried during early pregnancy, LH concentrations were significantly higher than in those whose pregnancy progressed.
Jacobs et al. (24) gave an explanation for this deleterious effect of high basal LH on successful pregnancy. Oocytes are normally held in the diplotene stage of their first meiotic division until immediately before ovulation. Meiosis has been shown to be arrested by an oocyte maturation inhibitor (OMI). This low molecular weight peptide is found within follicular fluid. The midcycle LH surge inhibits the production and/or action of OMI to allow meiosis to occur at the appropriate time. High basal concentration of LH during follicular development prior to the LH surge may prematurely initiate the resumption of meiosis resulting in the ovulation of post-mature oocytes. If fertilized, these oocytes may develop into embryos with a decreased chance of viability and early pregnancy loss.
Therefore, the controversy is centered upon the amount of LH necessary to support steroid production prior to ovulation and the role of LH in follicular maturation, ovulation and a successful pregnancy.
There is no debate, however, about the importance of the mid-cycle release of LH: it is a requirement for ovulation of a fertile ovum. This preovulatory surge of LH initiates not only events within the ova (resumption of meiosis) but also activation of enzymes that weaken the follicular wall to facilitate the extrusion of follicular contents.
Is RecLH necessary ?
Elevated levels of serum LH during the follicular phase of the menstrual cycle are not only unnecessary for follicular maturation but are deleterious to a normal reproductive process (6). These elevations may occur as a result of administration of exogenous LH or through an endogenous pathological process (ex. policystic ovary) (6).
The best results for ovulation induction would be expected with recombinant follicle stimulating hormone (RecFSH) administration to women following gonadotrophin releasing hormone (GnRH) down- regulation.
The re-evaluation of the two cell-two gonadotrophin theory suggests that during the preovulatory period, resting levels of LH are adequate for normal follicular maturation. Indeed, overstimulation of the ovary with excessive amounts of LH may diminish the ability of that target organ to produce fertile ova (13).
RecFSH induces growth of preovulatory follicles and this follicle growth does occur in the presence of subnormal estradiol level (7, 8, 11, 12).
LH is needed for adequate biosynthesis as a substrate for aromatase activity. This indicates that growth and steroidogenic granulosa cell activity may be differently regulated.
LH is important for the production of estrogens by stimulating the theca cells to produce androgens and then estradiol. The formation of LH receptors is induced by FSH in the granulosa cells of the dominant follicle and with this the importance for it (the follicle) to maintain its growth when FSH levels decline.
The follicles who have LH receptors are the only ones who are going to be able to respond to the LH pulse that activates the mechanisms that sparks ovulation.
In several studies of ovarian stimulation with RecFSH, normal follicular growth was induced but with low remaining levels of estradiol and androstenedione concentrations (7, 10, 11, 12).
This indicates that ovarian follicles are incapable of producing sufficient amounts of androstenedione (AD) in the presence of minute amounts of LH (bellow 0.38 IU/L) (10, 11, 12). The subsequent inability of normal estrogen production within follicles is in keeping with the two cell – two gonadotrophin hypothesis, indicating that FSH induced granulosa cell aromatase activity can only lead to augmented estradiol production if a sufficient amount of the aromatase substrate androstenedione is available (11, 15).
In order with this, stimulation experiments in rats with human recombinant FSH (hRecFSH) and human recombinant LH (hRecLH), resulted in increased ovarian estrogen secretion that only occurred if both hRecFSH and hRecLH were given simultaneously (2). Treatment with hRecFSH alone stimulated the granulosa cell aromatase activity without estrogen secretion, whereas hRecLH alone stimulated the thecal androgen synthesis and androgen secretion 2.
This underlines the concept of a LH threshold for sufficient estrogen production and for an optimal induction, thinking that exposure to hRecLH may improve embryo viability and the rate of development (3).
LH supplementation in the luteal phase may be necessary to sustain corpus luteum function in artificial cycles in which endogenous LH levels are suppressed (16). Progesterone production by the macaque and human corpus luteum during the menstrual cycle requires LH (17). Daily LH supplements after a bolus injection of u-LH in monkeys produced a luteal phase of elevated progesterone that was of normal length (18). Similarly, low doses of RecLH administered after ovulation induction maintained luteal function in a macaque (19).
The data suggest that RecLH could be a useful alternative to conventional hCG as an ovulatory agent in controlled ovarian stimulation cycles. With a shorter half life, RecLH may promote periovulatory maturation of a more select cohort of follicles, compared to a long-acting hCG bolus. This may be particularly relevant in women with a history of high risk ovarian hyperstimulation syndrome.
RecLH may be useful with RecFSH in follicular development paradigms and alone for luteal phase complementation.
It is hypothesized, based on in vitro observations 14, that physiologic FSH levels in combination with low local estradiol concentrations, as observed in several studies (7, 10, 11, 12), are insufficient for the induction of LH receptors and that these follicles are therefore not responsive to hCG, justifying the absence of a rise in serum progesterone.We have to mention that there are several potential non-physiologic effects of using hCG (16). These include:
Therefore , replacement with a more physiological stimulus, such as hRecLH, is an important consideration.
One of the problems of Ovarian Hyperstimulation Syndrome is the administration of the hCG dose to achieve ovulation.
Because new regimens of ovarian stimulation use the GnRH agonists for better results, we can not use them as a substitute for hCG, as proposed by Gonen and Imoedemhe (4, 5). This would be possible with the future introduction of GnRH antagonists for pituitary suppression.
Therefore another important consideration for the use of hRecLH would be for patients with high risk of Hyperstimulation Ovarian Syndrome.
The paper of inhibinHusueh et al. (25) published an important paper demonstrating that theca from the rat ovary responds to treatment with inhibin with increased production of androgen in vitro. Hilliet et al. (26) subsequently extended this finding to man, re-examined the action of inhibin on basal and LH responsive thecal androgen synthesis in vitro. Two important points emerged when inhibin actions on basal and LH responsive thecal androgen synthesis in vitro were examined (15):
These data are important for the interpretation of pure FSH action, since they imply that concomitant stimulation of the theca by LH might be necessary in order to achieve paracrine signaling induced by FSH to influence thecal androgen synthesis.
A new question arises for a marker of follicular development when using RecFSH because of the lack of LH and androgen secretion and having the estradiol/oocyte ratio as a strong index of in vitro fertilization success rate (1). Mitchel et al. (9) proposed to use inhibin (Alfa subunit) measured by radioimmunoassay, as its value was similar to estradiol as a marker for follicular development (9, 10).
For the majority of patients for whom FSH therapy is indicated, LH administration is not required to achieve follicular development as sufficient endogenous LH is present (as shown in women with WHO group II anovulation (28) and patients stimulated for Assisted Reproductive Technologies). In contrast, the majority of women with hypogonadotropic hypogonadism (WHO group I anovulation) do not have the threshold levels of endogenous LH required to achieve optimal follicular development and steroidogenesis during therapy with FSH alone. Among these women, urinary and RecFSH have been shown to achieve considerably lower estradiol levels than that obtained with HMG preparations containing both, FSH and LH (27). It also appears that in this population, the follicles stimulated by FSH alone do not consistently rupture after hCG administration. They luteinize poorly and oocytes may have a lower fertilization rate (27). An exogenous supply of LH is required to achieve an adequate follicular response.
Until recently, HMG, a urinary extract containing a fixed combination of LH and FSH, was the only source of exogenous LH for women with hypo-gonadotropic hypogonadism (HH). However, RecLH is now available for clinical use providing a new treatment option.
In a randomized trial "The European Recombinant Human LH Study Group" (28), demonstrated the clinical efficacy of hRecLH for supporting hRecFSH induced follicular development in women with HH. In a dose related manner, hRecLH promoted estradiol secretion, enhanced the effect of FSH on follicular growth and permitted successful luteinization of follicles when exposed to hCG.
The same study (28) also confirms when FSH alone is used to stimulate follicular development, follicular growth occurs, but estradiol secretion is minimal, resulting in deficient endometrial growth. In addition, when exposed to hCG, these follicles frequently fail to luteinize (27).
The dose of 75 IU of hRecLH for promoting optimal follicular development was sufficient in the majority of patients (28).
The successful induction of ovulation in women with HH and intact pituitary function has been achieved with pulsatile GnRh therapy. Pulsatile GnRh therapy has been the treatment of choice because it restores the pulsatile released gonadotrophins from the pituitary. This results in predominantly unifollicular cycles and satisfactory pregnancy rates. The treatment is associated with low rates of multiple pregnancy and is not complicated by ovarian hyperstimulation syndrome (29).
However, for patients who do not respond adequately to pulsatile GnRh or those with pituitary disease, HMG therapy administered as a once daily injection has been the only alternative treatment for ovulation induction.
Compared to HMG treatment for HH, the use of hRecLH offers a number of differences (28). 1. It is the first preparation of LH without FSH activity that is suitable and available for extensive clinical use. 2. It has a high specific activity suitable for s.c. injection, allowing self-administration by the patient. 3. By comparison, HMG preparations are given i.m. and contain a large proportion (~95%) of nonspecific copurified urinary proteins, which can cause hypersensitivity reactions. Furthermore, only once daily injection of hRecFSH is required in comparison with GnRh, which has to be administered every 60-120 min. 4. HMG is associated with an increased risk of multifollicular development, with potential complications of multiple pregnancy and ovarian hyperstimulation syndrome.
The development of hRecLH used in addition to hRecFSH provides another important therapeutic option for ovarian stimulation.
With recent findings in the use of hRecFSH and hRecLH, we could theoretical propose the use of hRecLH at the moment of sectorial follicle growth when LH receptors have been developed, to achieve a better control and a lesser amount of oocytes, and by titration of both gonadotrophin doses for individual patients. Because the two preparations are given separately, the dose of each gonadotrophin can be tailored to the individual's requirements to achieve the goal of unifollicular cycles.
The potential complications seen with HMG therapy (multiple pregnancy and ovarian hyperstimulation syndrome) may be reduced with hRecLH and hRecFSH therapy.
Imthurn et al. (30) suggested to administer 15,000 IU of hRecLH to mimic the mid-cycle LH surge. This practice would be affordable for very few patients because of the huge amount of hRecLH ampoules needed and that if we could produce unifollicular cycles it would be of no difference to the use of hCG as a substitute for the mid-cycle LH surge.
This hypothesis requires more investigation.
Edited by Aldo Campana,