The Role Of Luteinizing Hormone

Published Date: 02 Nov 2017

Authors: Dr Mamatha Deendayal, Dr Devika Gunasheela, Dr R Gutgutia, Dr Geeta Haripriya, Dr Mirudubhashini, Dr Nayana Patel, Dr Amit Patki, Dr Rahul Chavan*, Dr GAA Ramaraju**

* Co-author is employed by Merck Serono India.

** Corresponding Author

Disclaimer: This review article is the result of a series of advisory board meetings supported by Merck Serono.

Introduction

With the increase in studies on Luteinizing hormone (LH) and its mechanism of action on ovulation, oocyte maturation, and consequent fertilization and implantation, there has been an ever increasing confusion. FSH and LH have striking similarities not only in terms of the organ of secretion of the two hormones, their action on the same target organ, producing similar response, but also the molecular structure of the two hormones which is 80% similar and act at similar receptors. [1,2]

The most commonly used protocol in assisted reproductive technology (ART) consists of controlled ovarian hyper-stimulation (COH) with daily injections of recombinant human Follicle stimulating hormone (r-HFSH) to induce multiple follicle growth in the ovaries along with daily injections of a gonadotropin-releasing hormone (GnRH) agonist or antagonist to prevent premature LH surge and premature ovulation. The pituitary down-regulation (endogenous pituitary suppression) that is achieved with GnRH analogues creates an environment where LH is deficient or very low, and which may be detrimental to the development of normal healthy follicles. It has been shown that growing follicles become increasingly sensitive to, and ultimately dependent on, the presence of LH for their development. [3]

Documented results associate poorer outcomes with patients whose LH concentration was low, after pituitary suppression was achieved with GnRH analogue treatment. [4, 5]

Some assisted reproduction practitioners thus advocate add-back LH to the mid-follicular phase in ovarian stimulation cycles. Other practitioners deem add-back LH to be unnecessary, justifying that the small amounts of LH present after down-regulation are sufficient to sustain theca and granulosa cell stimulation. [6]

Cycles with recombinant LH supplementation have shown lower levels of cumulous cell apoptosis than FSH-only cycles, possibly indicating improved oocyte quality in LH-supplemented cycles. [7]. Also Lower follicular fluid vascular endothelial growth factor (VEGF) levels in the rhLH group might be responsible for less granulosa cell apoptosis in the rhLH group. [8]

It is possible that the administration of LH restores the follicular mileu of the developing follicle in older ART patients. [9]

Quite a few predictors of poor outcome of COH have been identified, namely age (above 35 years) poor ovarian reserve, poor responder to previous ART cycles, hormonal status (FSH, LH, estradiol, anti-mullerian hormone) and genetic variations.[6]

Identification of gene structure for gonadotropins receptors and subunits has led to better understanding of the normal and pathological functioning of hypothalamic-pituitary-gonadal axis. [10]

Genetic variability also seems to be an important factor in determining the ovarian response in COH and IVF. With the recent increase in interest in LH pleomorphism, it has been found that there are a multitude of genetic variations of the LH receptor (LHr) gene. These variations when present at a single nucleotide protein site are called snp pleomorphism. There is also evidence on the presence of activating and inactivating mutations of LH receptor, apart from a common polymorphism in LHβ subunit gene, which could lead to pathologies related to LH-dependent gonadal functions. [10, 11]

Although recent researches have facilitated better understanding of the LH and FSH hormone inter-relation and effect on fertilization and implantation, there is still confusion on the usefulness of its supplementation in ART cycles.

The use of rLH in ART should be guided by a rational that is based on the need of the patient. To combat this confusion it is essential that we understand the crux of the matter based on the studies done in recent times and the ramifications of those studies in terms of better defining the patient subpopulation that will benefit with rLH therapy in ART.

Physiological hormonal interplay:

As per the two-cell, two-gonadotropin theory, FSH is necessary for stimulating antral follicle and follicular growth, while LH plays a role in stimulation of androgen secretion by thecal cells in the pre-antral stage. The LH and FSH synergism is thus vital for steroidogenesis and subsequent follicular ability to ovulate and luteinize on exposure to mid-cycle LH surge. [1, 2, 12]

"Two-cell Two-gonadotropin" theory: [1, 2]

The ovary comprises of two cellular components, which are stimulated independently by LH and FSH, leading to the production of ovarian steroids. [13] The theca cell segment of the follicle is stimulated by LH, which facilitates androgen production from cholesterol, while FSH plays a role in converting androgen precursors to estrogens in the granulosa cell compartment. [14] The figure 1 given below explains the hypothesis.

Figure 1: Two-cell Two-gonadotropin theory

Ovarian steroidogenesis in the preovulatory follicle takes place through LH receptors on theca cells and FSH (possibly also LH) receptors on granulosa cells (see Fig. 1). The steroidogenic acute regulatory protein (StAR protein) is the primary regulator of production of androstenedione, which subsequently diffuses into granulosa cells to serve as the estrogen precursor. [1, 2]

In the preovulatory follicle, cholesterol in theca cells arises from circulating lipoproteins and de novo biosynthesis. FSH is responsible for follicular growth and estrogen formation. FSH induces preovulatory granulosa cells to form estradiol primarily through aromatization of androstenedione. This androstenedione serves as the estrogen precursor in the granulosa-lutein cells. [1, 2]

In the corpus luteum, large deposits of cholesterol arise primarily from circulating lipoproteins to support the production of extremely high quantities of progesterone. Theca-lutein cells possess LH receptors and produce androstenedione. [1, 2]

The granulosa-lutein cell of the corpus luteum are luteinized and heavily vascularized and contain large quantities of cholesterol, they also contain high levels of LH receptors in addition to FSH receptors and they produce large quantities of progesterone that is regulated primarily by LH and StAR. Granulosa-lutein cells also aromatize androstenedione of thecal origin and eventually give rise to estradiol formation. [1, 2]

Concept of FSH threshold:

The concept of the FSH ‘‘threshold’’ proposed by Brown postulated that in gonadotropin therapy, the ovary has a minimum requirement level (threshold requirement) for FSH below which follicular development does not occur. [15, 16]

Increasing the dose by a factor of only 10–30 % above this threshold causes normal rates of follicular stimulation, and FSH levels above this minimum level increases the chances of excessive stimulation. So this additional increase over the threshold level causes multiple follicular growths, a higher incidence of multiple pregnancies and OHSS. The duration of the period in which the threshold is exceeded (the FSH ‘‘window’’) has a crucial role in the follicular recruitment. [17, 18]

More recent studies also confirm that follicular growth does not occur below the threshold levels. [19]

Figure: 2 FSH Threshold and Recruitment Window

Following optimum FSH stimulation, there is follicular recruitment, growth, selection and dominance. Subsequent development of this cohort during the follicular phase becomes dependent on continued stimulation by gonadotropins. Increasing FSH concentrations should surpass the threshold level to initiate the final gonadotropin-dependent phase of follicular growth. [20]

There is secretion of increasing amounts of estradiol during this phase. The peripheral estradiol levels are increased with feedback inhibition of FSH secretion. The maturing follicle inhibits FSH secretion leading to a fall in its levels below threshold, thus stopping less mature follicles from maturing. [21, 22]

Further, it has been shown that FSH threshold is not fixed for any given follicle, but depends on developmental stage and varies over time. [23] The follicles exhibit different degrees of FSH sensitivity at the time of recruitment; highest need for FSH is at the early antral stage, and declines in the late antral stage. The follicle with the highest sensitivity will benefit most from increasing FSH levels and will subsequently gain dominance. [24]

The suggested reasons for the response of ovarian follicles to certain FSH level than to a specific dose are fluctuating levels of the endogenous production of gonadotropin,[15] long half-life of FSH preparations, and up regulation of its receptors due to FSH administration. [19]

Although FSH can induce follicular growth even without LH, there is evidence that the follicles may have developmental deficiencies like abnormally reduced estradiol production, and lack of ability to luteinize and rupture following hCG stimulus. [24] Hence, a certain amount of LH exposure is necessary for optimal follicular development.

Another possibility is that FSH stimulates the production of Progesterone (P) by driving cholesterol conversion into the steroid pathway.[25-29] Early increased exposure to P can advance the endometrium, leading to asynchrony of embryo development to endometrial development and reduction of implantation. LH stimulates the conversion of Progesterone (P) into androgens, which can be further aromatized to estrogens (E). The addition of LH may benefit the endometrium by decreasing the risk of a premature P increase and therefore improve the likelihood of implantation and clinical pregnancy. [28, 29]

Concept of LH therapeutic window:

The concept of LH therapeutic window was introduced in ART to facilitate ovulation induction and successful conception by facilitating the clinician to administer precise and consistent dose of gonadotropin in those who require exogenous LH, without the risk of overdosing. [30] The concept of LH therapeutic window has been explained in brief in Figure 3.

Figure 3: LH therapeutic window

The usefulness of exogenous LH in ovarian stimulation protocols in assisted reproduction continues to be a matter of debate.[6] Studies have shown that serum LH levels should be between 1.2 IU/L and 5.0 IU/L [31] for optimal development follicle in cycles where endogenous LH is suppressed. [6, 24]

Though there are no clear guidelines on when exactly LH needs to be added, some of the recent studies done suggest that the indicators for adding LH to a ART cycle are mid follicular (day 6) hypo-response on long GnRH agonist, no follicles > 10mm, E2 < 200 pg/ml, endometrial thickness < 6mm and baseline serum LH < 1.2 IU/ml on day 6. [31, 32]

A recent meta-analysis done by Hill et all on the use of LH in art in advanced patient age group concluded that of a total of 7 RCT’s, 5 were in favour of adding LH in ART therapy in patients of advanced age group as against 2 which did not advocate the same.[33]

However it is critical that the Ad-back LH should be done in appropriate patients as excess of LH going above the ceiling will cause suppression of granulose cells and follicular atresia. [6, 33]

Pharmacogenomics and Ovarian stimulation:

FSH Polymorphism:

Patients with unfavourable FSHR genotypes have been shown to need higher doses of rFSH to overcome relative ovarian insensitivity. FSHR gene may have an important role in the success of ovarian stimulation. Women with the 307 Ala and 680Ser are associated with reduced COH outcomes. 680Ser is specifically associated with lower clinical pregnancy. These patients when undergoing IVF treatment are characterized by higher basal FSH serum concentrations, higher administered amounts of FSH required and higher risks of hypo- or hyper-responses. Up to 35% of IVF patients are detected with alternatively spliced FSHR products. Genotyping the FSHR Asn680Ser SNP, together with some additional novel markers (e.g. transcript levels), may therefore provide a means of identifying a group of poor responders before infertility treatment is initiated. [34, 35]

LH polymorphism:

The LH receptor gene is known to carry many single nucleotide polymorphisms (SNPs), with an estimation of 282 SNPs.[36] In 1991, Pettersson and Söderholm identified a common genetic LHβ variant or v-βLH owing to the alterations in two polymorphic base changes in the β subunit gene leading to changes in the amino acid sequence, Trp8Arg and Ile15Thr. They had initially suggested this discovery as an immunological anomalous LH form. [37, 38]

The shorter half-life of v-βLH may be linked to the presence of extraglycosylation signal into the β subunit that could lead to an addition of second oligosaccharide to Asn13 of the β protein. It has been found that there is more potency of the overall LH activity of v-βLH at the receptor site; however, its duration is shorter in vivo. [39]

Determining the impact of this variant on reproductive health, many trials have noted its association with ovulatory disorders, premature ovarian failure, hyperprolactinemia, luteal insufficiency, menstrual disorders, endometriosis, and infertility. [40] An observational study noted low response in some women following ovarian stimulation, resulting in greater need for rFSH (>2500 IU). [39]

Apart from variations in LH receptor, polymorphisms could also occur in genes regulating estrogen biosynthesis, FSH signaling, and folate metabolism, which affects response to ovarian stimulation, leading to changed IVF outcomes. [41]

Earlier studies have demonstrated that subjects with poor COH response requiring increased rFSH may show improved outcomes with LH supplementation. Further research is thus paramount to confirm if such polymorphisms could be markers of COH outcomes, which may in turn identify those patients who would benefit from LH supplementation. [39]

Optimising FSH dosing:

Various studies suggest four parameters of FSH administration management involved in the risk of multifollicular development: (a) the choice of the FSH starting dose, [42, 43] (b) The duration of the starting dose before stepping up or stepping down, [42, 44] (c) the rate of increase in FSH dose at each increment [45] and (d) the reduction of the FSH dose once a follicle has been selected. [46]

In an attempt to prevent the risks of overstimulation and multiple pregnancies it is crucial to use a low starting dose of FSH [45], and to use small increments in the daily dosage [42,44,45].

Exogenous LH supplementation:

Luteinizing hormone is important in regulating steroidogenesis throughout follicular development; adequate LH is particularly important for oocyte maturation. [47]

Most of the Asian assisted reproduction practitioners make use of both long agonist and antagonist protocols for ovarian stimulation; experience with the former is greater. Published literature on the beneficial effects of exogenous LH in patients with previous suboptimal response or low baseline serum LH concentrations is more extensive in long agonist protocols (Lisi et al., 2003; De Placido et al., 2004, 2005; Franco et al., 2009; Pezzuto et al., 2010). Documented results associate poorer outcomes with patients whose LH concentration was low after gonadotrophin-releasing hormone (GnRH) agonist treatment. [48, 49]

The Asia Pacific Fertility Advisory Group [6] in 2011 strongly recommended rLH co-treatment with rFSH in patients with history of poor response

suboptimal response on day 6 in long agonist cycles

absence of >10 mm follicles

endometrial thickness of <6 mm

estradiol levels <200 pg/mL

rLH may also be beneficial in women aged >35 years undergoing ovarian stimulation with long-agonist or -antagonist protocols.[6]

Poor responders and low ovarian reserve:

Many factors are linked to decreased ovarian response and hence it is difficult to identify poor responders. Although several tests have been suggested, none can indicate it accurately. [50]

Some putative biomarkers to identify poor responders includes : (i) LH concentrations either at baseline or day 6 midfollicular –and (ii) anti-Mullerian hormone (AMH)levels (iii) antral follicle count (AFC).

Wong et al recommend that further research needs to be done in patients with suboptimal response based on the following biomarkers: (i) AFC <6 in both ovaries; (ii) AMH concentration <1.5 ng/ml; and (iii) LH polymorphisms. [6]

Poor ovarian reserve is estimated to occur in around 26% of the IVF cycles. Evidence indicates that rLH administration along with rFSH in such patients may help in improving ongoing pregnancy rates in poor responders and advanced aged women. [51-55]

On the contrary, Barrenetxea et al, 2008[56] and a more recent study (Bosdou et al, 2011) [57] concluded that there is insufficient evidence to validate the effectiveness of rLH in subjects with poor response, undergoing IVF cycles.

LH polymorphism:

In another preliminary study, the total rFSH consumption was elevated during ovarian stimulation due to the presence of v-βLH [37]. Based on the findings, the researchers indicated the potential of v-βLH as a marker of ovarian responsiveness to rFSH. This role of v-βLH, if validated with further research, could thus facilitate clinicians in identifying patients requiring exogenous LH addition during ovarian stimulation. [39]

Advanced reproductive aged patients:

A recent systemic review and meta-analysis (Hill et al, 2012,)[52] concluded that the inclusion of rLH to FSH stimulation enhanced the clinical pregnancy and implantation rates in ART cycles in patients aged ≥35 years. Similar results were reported in many other randomized trials. [54, 55]

An open-label randomized controlled study (Bosch et al, 2011)[55] found that rLH is beneficial in improving the implantation rate in women aged 36 to 39 years, but not so in those younger than 36 years of age. [55]

This is due to the fact that the serum androgen levels decline steeply with age, as does there response to FSH stimulation. LH administration enhances follicular androgen production and followed by aromatization to estrogen. It also controls progesterone production by granulosa cells which is also FSH dependent. Thus LH supplementation seems appropriate for poor responders where it restored the follicular and endometrial milieu and improves the cycle outcome. [58, 59]

Another retrospective observational study evaluating ART patients undergoing stimulation with an antagonist procedure in a Boston IVF centre, reported clinical pregnancy success of 36.0% for patients aged 38 years treated with rFSH and rhLH compared with 19.1% (p = 0.048) for those stimulated with rFSH and HMG.[60]

Polycystic ovary syndrome (PCOS):

The detrimental impact of endocrinological disorder, which is linked to hyper-secretion of LH and ovulatory dysfunction, is attributed to increased LH levels. Studies have found that such women are associated with poor fertilization, oocyte quality, and embryo quality, which could be due to underlying mechanisms such as androgen excess induced by LH. However, contrary to previous belief, it was later demonstrated that hyper-insulinemia and not LH hyper-secretion plays a vital role in PCOS pathogenesis.[61] Adding LH in this scenario would lead to OHSS and hence LH shold be avoided.

Role of LH in ART:

LH supplementation is prime in older and poor-responding patients because they usually receive higher FSH doses for COS, they show higher P levels at the end of stimulation, and, subsequently, their endometrium receptivity diminishes.[62]

Moreover, GnRH antagonists are used currently and mainly in older patients and in those exhibiting a low ovarian response to the GnRH long agonist protocol.[63]

Dosing of LH:

In 1998, the European Study Group conducted the first randomized efficacy clinical study to investigate the safety and tolerability of rLH supplementation in hypogonadotropic hypogonadal women (WHO group 1 anovulation). The researchers also aimed to assess the minimal effective dose for this patient population. The patients (n=38) randomly received daily injections of 0, 25, 75, or 225 IU of rhLH in conjunction with 150 IU rhFSH/day for up to 20 days. The results showed that rhLH helped in:

Promoting dose-associated increase in the secretion of estradiol and androstenedione by rhFSH-induced follicles

Enhancing ovarian sensitivity to FSH as observed by the number of patients who developed follicles following FSH administration

Increasing the successful luteinization of follicles on exposure to hCG

It was observed that 75 IU rLH promoted adequate follicular development and steriodogenesis in 46% of the treatment cycles, with sufficient secretion of estrogen and progesterone in 75-80% of the cycles. Based on the findings, the researchers recommended that 75 IU rLH is effective in most of the women by facilitating maximal endometrial growth and optimal follicular development, which is defined as:

≥1 follicle of ≥17 mm

Estradiol levels of ≥400 pmol/L

Mid-luteal phase progesterone level of ≥25 nmol/L

Furthermore, they suggested that small percentage of women may require up to 225 IU of rLH/day subcutaneously, but emphasized that the high dose of rLH was also found to be immunogenic and well-tolerated.[64]

To achieve an optimal benefit Ramaraju et al also suggest a dose of 75 IU/day of rLH for supplementation with rFSH. [32]

Regarding the dosage, a ratio of 2:1 for FSH:LH , i.e., 150IU:75IU starting on day 1 or 6 of stimulation is commonly used, especially in hypo-hypo patients (Wong et al, 2012).[6] Several studies have indicated the beneficial effect of LH in women aged >35 years in agonist and antagonist protocols.[55] Similarly, a Cochrane review reiterated the usefulness of rLH in poor responders and advanced aged women at risk of spontaneous miscarriage.[53]

A study done by Lisi et al, showed that the administration of rLH (75 IU/day, for 4 days), one day before the beginning rFSH stimulation, offers some benefits in terms of clinical pregnancies when compared with the patients undergoing stimulation with rFSH alone.[65]

Though starting patients with rLH on day 1 maximizes the benefit of increased ovarian androgen production triggered due to the presence of the exogenous LH, it will act synergistically with FSH to promote FSH receptor mRNA expression, follicular development and steroidogenesis (Weil et al., 1999).[66]

Studies have also fortified that rLH in combination with FSH is better than HMG with FSH. This might be due to excessive or inconsistent LH activity from the hCG component in HMG may affect ocyte maturation in the latter half of the ovarian stimulation cycle, giving rise to the differences in numbers of oocytes retrieved and success of pregnancy. [67, 68]

Conclusion:

Optimal follicle development with subsequent ovulation requires the complex interaction of FSH, LH, and their complementary activities. The ART outcome can be improved with optimisation of FSH dose in various patient populations and supplementation of LH in various subgroups discussed above. Biomarkers to ascertain women who are in need of exogenous LH need to be sought. With the increasing evidence of pharmacogenetic approaches it is likely that choice of ART regime will be also guided by patient’s genetic makeup. So despite of an increasing interest on the use of adjuvant LH with FSH in ART cycles, no clear consensus has been reached on dosage, timing, and patient population who would benefit the most with the treatment.