Two Hormones for One Receptor: Evolution, Biochemistry, Actions, and Pathophysiology of LH and hCG

LH and chorionic gonadotropin (CG) are glycoproteins fundamental to sexual development and reproduction. Because they act on the same receptor (LHCGR), the general consensus has been that LH and human CG (hCG) are equivalent. However, separate evolution of LH b and hCG b subunits occurred in primates, resulting in two molecules sharing ~85% identity and regulating di ﬀ erent physiological events. Pituitary, pulsatile LH production results in an ~90-minute half-life molecule targeting the gonads to regulate gametogenesis and androgen synthesis. Trophoblast hCG, the “ pregnancy hormone, ” exists in several isoforms and glycosylation variants with long half-lives (hours) and angiogenic potential and acts on luteinized ovarian cells as progestational. The di ﬀ erent molecular features of LH and hCG lead to hormone-speci ﬁ c LHCGR binding and intracellular signaling cascades. In ovarian cells, LH action is preferentially exerted through kinases, phosphorylated extracellular-regulated kinase 1/2 (pERK1/2) and phosphorylated AKT (also known as protein kinase B), resulting in irreplaceable proliferative/antiapoptotic signals and partial agonism on progesterone production in vitro . In contrast, hCG displays notable cAMP/protein kinase A (PKA)-mediated steroidogenic and proapoptotic potential, which is masked by estrogen action in vivo . In vitro data have been con ﬁ rmed by a large data set from assisted reproduction, because the steroidogenic potential of hCG positively a ﬀ ects the number of retrieved oocytes, and LH a ﬀ ects the pregnancy rate (per oocyte number). Leydig cell in vitro exposure to hCG results in qualitatively similar cAMP/PKA and pERK1/2 activation compared with LH and testosterone. The supposed equivalence of LH and hCG has been disproved by such data, highlighting their sex-speci ﬁ c functions and thus deeming it an oversight caused by incomplete understanding of clinical data. (Endocrine Reviews 39: 549

L H and the primate-specific chorionic gonado- tropin (CG) are glycoproteins fundamental for sexual development and reproduction.Both hormones have long been considered equivalent, because they bind the same receptor, the LH chorionic gonadotropin receptor (LHCGR) (), which is mainly expressed in the gonads, and similarly activate the classical cAMP/protein kinase A (PKA) steroidogenic pathway.In clinical practice, human CG (hCG) is the hormone of choice when LH activity is needed (e.g., in the treatment of hypogonadotropic hypogonadism (HH) or in assisted reproduction technologies (ARTs), because it is easily purified in high concentration from urine of pregnant women.Historically, human LH of pituitary origin was difficult to obtain and lacked full biological activity because it embeds a proteolytic site leading to internally cleaved hormones and thus displays one half of the activity of the intact molecule (, ).Human recombinant LH only recently became available for clinical use in ARTs.However, the experience is insufficient to draw conclusions about different, specific indications for LH and hCG in clinical practice.Thus, the idea that human LH and hCG might be used indifferently remains dogmatic.However, because evolution led to the appearance of several copies of the CGB gene in primates, the equivalence between the two gonadotropins with "LH activity," interacting with the single receptor, requires a critical reassessment.Physiological considerations suggest that LH is unique and fundamental for gametogenesis regulation.In contrast, the evolutionary onset of CG genes might be linked to the requirement for different signals specifically supporting pregnancy in primates.Modern technologies and recent in vitro data have allowed for the evaluation of multiple signaling pathways, activated by a number of LHCGR interactors (, ).A recent report has suggested that a complete picture of LH and hCG action might be missing when exclusively investigating the classical cAMP/PKA steroidogenic pathway ().In vivo and in vitro studies using rodents or rodent-derived cells have provided informative results in understanding the physiology of gonadotropins (, ).However, because rodents do not have CGB genes, they do not produce CG, and the murine LH receptor is not identical to the human receptor (% of the identity between Rattus norvegicus Lhr and LHCGR).Some recent evidence has suggested that the human LHCGR possesses a specific region capable of distinguishing between LH and hCG ().These studies raise the question of whether hCG and LH actually are equivalent and fully interchangeable in humans.In addition, because rodents are still being used to calibrate the gonadotropin preparations used in medical practice () and the physiology of LH action was mainly obtained from murine models, another question is whether the primate-specific nature of the dual ligand system provided by LH and CG has been completely characterized.Some clinical () and in vitro (, ) comparisons of commercial LH and hCG preparations have been performed in the past  years, revealing the existence of several peculiarities but leaving many unanswered questions.
We have reviewed the current evidence available on the similar and different functions of LH and hCG.
Two Ligands for One Receptor: Why?
Ligands and receptors evolved along with their molecular targets, resulting in exclusive, hormone-specific regulation of pathways and physiological functions ().In primates, the existence of LH and hCG as ligands for the same receptor suggests that a separation of hormone-specific roles occurred, resulting in different, hormone-specific physiological functions.These distinct roles might exist to fulfill different requirements for the regulation of fetal development (in females) and gametogenesis (in both sexes).These reproductive functions are accompanied by the prevalent-or even exclusive-time-and sexdependent presence of only one of the two hormones, pointing toward specific physiological targets reasonably regulated by different endocrine signals ().LH-and hCG-specific actions exerted at the molecular level are underpinned by different molecular structures associated with each hormone (), which arise from a different set of related genes (), along with source cell-dependent posttranslational modifications ().Although previously considered "equivalent," both hormones divergently evolved in primates and are characterized by differences at the genetic, molecular, and physiological level.
Although the specificity of LH and hCG signals has not been completely elucidated, it is supported by the different nature of their target cells.In women of fertile age, LH exerts its best-known functions in the ovary, where it mediates proliferative signals in the granulosa cells coexpressing the FSH receptor (FSHR) and LHCGR and stimulates androgen synthesis, mainly androstenedione, in theca cells exclusively expressing LHCGR.Moreover, LH induces luteinization of granulosa cells, progesterone synthesis, and corpus luteum maintenance during the luteal phase of the menstrual cycle.In males, Leydig cells in the testis are targeted by LH, which induces testosterone production as the major synthesized androgen.LH is therefore essential for reproduction in both sexes.In contrast, hCG physiological action is only exerted in females as a massive progesterone stimulator in the corpus luteum and mediating placental growth during pregnancy; it is not produced in males.These considerations suggest a sex-specific role for each hormone in humans and other primates.Such roles are quite different (gametogenesis vs pregnancy) and most

ESSENTIAL POINTS
• In the past decade, the two hormones LH and human chorionic gonadotropin (hCG) were considered equivalent because they bind the same receptor, clearly activating the classically known cAMP/PKA steroidogenic pathway • Clinical evidence of small or undetectable different outcomes between LH and hCG usage has underlined this concept • Recent in vitro studies have demonstrated that intracellular signaling, downstream events, and cell fate are specifically mediated by LH and hCG • LH preferentially activates ERK1/2-and AKT-dependent proliferative signals, and hCG is mainly progestational, supporting the physiological roles of the two hormones • In the past 20 years, studies comparing the use of commercial LH and hCG preparations in reproductive medicine have provided clinical evidence of the differences observed in vitro, confirming the in vitro results • These data indicate that LH and hCG have unreplaceable roles, overthrowing the previous concept that they are equivalent and revisiting the basis on which clinicians decide on the application of these hormones likely not fully interchangeable.Clinical experience has shown that steroidogenesis and gametogenesis can be supported by hCG administration in both sexes.However, is human (primate) pregnancy sustainable by LH in the absence of hCG?In assisted reproduction, hCG is administered to provide LH-like activity based on the androgen-stimulating potential of the molecule.However, is the action of both molecules on gamete maturation identical?Compelling results were provided by a comparison between LH and hCG administered to mouse oocytes cultures at the germinal vesicle stage in vitro.A greater maturation rate after hCG treatment was revealed, and LH positively affected early embryonic development ().These data, albeit obtained using mouse tissues, suggest that different signals are mediated by each ligand, resulting in different effects on the physiological target.This is but one example of a number of recent in vitro and clinical studies providing a novel, unexpected view of LH-and hCG-specific roles.In the next paragraphs, we define such different roles starting from evolutionary considerations.

Phylogenies and Evolution of LH and hCG
The molecular structures of gonadotropins and their receptors are, overall, conserved during evolution and share similarities with several other ligands and receptors across the phylogenic tree.This hints at a common ancestral origin of these molecules and the promiscuity of molecular mechanisms involved in endocrine regulation.Gonadotropins are glycoprotein hormones belonging to the superfamily of cystine knot growth factors.Members of the cystine knot growth factor group share an arrangement of six disulfidelinked cysteine residues that achieve a structurally related "knot" conformation, despite a relatively low sequence homology ().Glycoprotein hormones possess a common a and a specific b subunit, assembled to form a noncovalently linked heterodimer () acting on specific leucine-rich repeats (LRRs) and rhodopsin-like G protein-coupled receptors (GPCRs).It has been estimated that the evolutionary origin of glycoprotein hormones and their receptors occurred at the origin of the metazoans.Hence, they organize the regulation of a wide range of endocrine systems and reproductive and metabolic functions that differentiated during evolution ().Such evolutionary issues were inferred by evaluating the genetic structure of gonadotropins and their receptor-encoding genes.A number of glycoprotein hormones (i.e., gonadotropins) and receptor variants developed as an endocrine adaptation to specific environmental conditions and the physiological changes required to improve reproductive success (fitness) tested by natural selection ().For example, the fertile window length, menopausal age, and high pregnancy success might be the result of species-specific, or even individual-specific, optimizations of reproduction implemented to best balance fitness and selective pressure.In primates, the appearance of choriogonadotropic hormones acting as additional ligands for one receptor suggests that different levels of regulation are required to manage gametogenesis and pregnancy.
Glycoprotein hormones across the phylogenic tree Glycoprotein hormones and their receptors revealed common structural folds, suggesting a common evolutionary origin of ligand-receptor pairs, likely resulting in similar and even promiscuous binding and signaling mechanisms.Binding specificity would be driven by the protein sequence spanning between the th and th cysteine residues of the hormone, consisting of similar sequences and falling within the LRR domains of their receptors ().In insects, the only molecule related to vertebrate glycoprotein hormones is the bursicon hormone, an approximately -kDa protein displaying a cystine knot heterodimeric structure and proapoptotic activity at the epidermal level ().The analysis of the bursicon hormone-encoding DNA sequence revealed high identity with glycoprotein hormone-like peptides found in silkworms, sea urchins, jellyfish, and corals, suggesting their evolutionary proximity ().Similar homologies were found by comparing the DNA sequences encoding the hormone receptors, demonstrating that the ligand-receptor system underwent coevolution.Coexpression of the LHCGR gene has been coupled to the canonical cAMP pathways in transgenic Drosophila melanogaster, similar to that in humans, further supporting conservation of the intracellular machinery necessary for GPCR signaling in invertebrates ().An evolutionary step toward vertebrates might be provided by a glycoprotein hormone of adenohypophyseal origin found in hagfish.This hormone consists of bound a and b subunits and the presence of their mRNAs matches the developmental stages of the gonad (), likely providing the earliest pituitary-gonadal system in vertebrates.In basal vertebrates, such as the hagfish, unique glycoprotein hormones regulate all the cognate gonadotropin functions found in mammals by acting on distinct receptors (fshr and lhr) mediating gametogenesis ().This ancestral form of glycoprotein hormone, homologs of which are conserved in vertebrates and some invertebrates, is known as thyrostimulin and is capable of increasing intracellular cAMP in cell systems expressing lamprey glycoprotein hormone receptor ().The evolutionary history of more physiologically specialized gonadotropins started ~ million years ago, when the ancestral gene encoding the thyrostimulin b subunit repeatedly duplicated, resulting in an LHb-encoding gene from which the TSHb-and FSHb-subunitencoding genes subsequently originated ().Further molecular specializations occurred in primates and equids, in which CGb and CGb-like LHb molecules, respectively, developed as gonadotropins of pregnancy.
Coevolution of glycoprotein ligands and their receptors testifies to the specificity of the glycoproteinmediated signals demonstrated in cartilaginous fishes, providing an ancient representation of the current diversity of reptilian, avian, and mammalian endocrine systems ().However, endocrine signal promiscuity is retained in some organisms such as zebrafish, where fsh binds both fshr and lhr, and only lh is specific for its own receptor ().As a consequence of this incomplete functional differentiation, the endocrine control of spermatogenesis in zebrafish relies on fshr expression in both Leydig and Sertoli cells ().Fish gonadotropin receptors might be activated by mammal cognate ligands and vice versa, suggesting a limited number of specific binding residues were conserved during evolution, even in the case of hCG binding to fish lhr ().The sharing of interspecific binding capability between gonadotropins and their receptors is maintained across fish and mammals as a legacy of common ancestral evolution.Moving from invertebrates to primates, however, the increasing complexity and specificity of the glycoprotein hormones and receptors is the requisite of the refined function of the pituitary-gonadal axis.The result is increasing endocrine specialization with species evolution.

LHB gene duplication: appearance of choriogonadotropin
LHb and CGb subunits are encoded by a cluster of tandem genes, located in humans on chromosome q.,which embeds a total of eight genes and pseudogenes ().There are six transcriptionally active CGB genes () and related promoters ().The LHB/ CGB gene cluster reaches the highest complexity in Homo sapiens, and other primates feature a simpler organization characterized by fewer CGB genes.Because most of the % difference between humans and chimpanzees is due to genomic insertions and deletions (), CGB genes putatively evolved by repeated duplications of an ancestral LHB gene common to all species of the primate lineage to meet specific physiological requirements (, ).High crossover activity putatively occurred in the human LHB/CGB cluster and resulted in sequence inversions generating palindromic genes ().One of the most accredited theories explains the rise of CGB genes and the increasing glycosylation rate of CGb molecules as evolutionary adaptations to the elevated energy demands for fetal development.The resulting highly specialized regulation of angiogenetic signals and myometrial invasion is necessary to support hemochorial placentation in humans and higher primates ().Thus, six CGB genes are found in humans, four to five in great apes, and one to three among Macaca, Callicebus, and Aotus ().The highly conserved CGB and CGB gene sequences in humans and great apes suggest they could have relevance for implantation and placental development in higher primates ().Other CGB genes (i.e., CGB, CGB, CGB, and CGB) are recognized as a source of hCG products during human pregnancy ().Accordingly, simpler primates (e.g., strepsirrhine) and other mammals have only one LHB gene and feature epitheliochorial placentation characterized by the presence of both the uterine epithelium and the maternal vascular endothelium during pregnancy.The theory explaining the rise of CGB genes has recently been extended ().Sexspecific functions (i.e., placentation, folliculogenesis, and ovulation in females and spermatogenesis in males) would be driven by the expression of sexspecific genes belonging to the same genomic cluster.In this context, the LHB gene is relegated to maintenance of physiological functions common to both males and females, because they could require similar regulation at the intracellular level, and CGB genes permitted independent evolution of signals specifically required for placentation in females, as an example of sexual dimorphism.This strategy might have arisen to solve an intralocus sexual conflict, which occurs when traits encoded by the same locus lead to conflicting fitness outcomes between the two sexes ().However, the evolution of placentation in primates might be linked to the gene encoding the glycoprotein hormone a subunit (common glycoprotein a, CGA) ().This gene has two splice variants, one of which is only found in anthropoid primates with the exonization of an Alu sequence.The additional encoding sequence results in an N-terminal extension improving protein stability.Similarly, CGB genes are characterized by an additional DNA sequence compared with LHB.Although a certain grade of similarity is shared among genes encoding glycoprotein hormone b subunits, CGBs display a peculiar extension of ~ nucleotides compatible with  amino acids at the C-terminal region ().This C-terminal extension (CTP) could have originated from the loss of the stop codon, occurring together with the duplication of an ancestral LHB gene, resulting in inclusion of the 9-untranslated region within the protein coding sequence, which is absent in all known mammalian LHB genes, except for equids (Table ).The human CTP domain contains four potential O-linked glycosylation sites, is enriched in serine, threonine, and proline residues (), and can be targeted by specific antibodies owing to the immunological potential of the glycosylated sites ().

Classical views of LH and hCG physiology
Although the diversification of endocrine axes and the appearance of CG molecules suggest that different LH receptor-dependent intracellular functions are required to regulate gametogenesis and pregnancy, experimental and clinical observations in women and female primates failed to clearly distinguish the actions of these molecules during gametogenesis () and luteal regression ().Thus, it has been widely accepted that these two molecules are equivalent.However, the evaluation of gonadotropin-specific functions in human physiology points to distinct, cell-specific roles for each molecule.In ovarian granulosa cells, proliferative signals directly delivered by LH are not necessarily exerted through the synthesis of androgens, which instead are the main product of LH action in theca cells.In contrast, hCG naturally only replaces LH in the luteal phase and beyond, during pregnancy, when steroid production is mainly limited to progesterone.
In humans, gametogenesis progresses owing to an orchestrated regulation by FSH, LH, growth factors, and steroid hormones.In women, the ovarian follicle is the functional unit deputed to oocyte maturation and growth, characterized by a dynamic structure of somatic cells surrounding the gamete ().Folliculogenesis starts ~ weeks after conception, when the follicular population comprises four to seven million oocytes at the resting primordial stage.Pools of these follicles progressively mature by undergoing morphological and molecular changes and passing through the primary and secondary gonadotropinindependent stages.Subsequently, from pubertal onset, monthly recruitment is guided by the expression of FSHR and LHCGR at relatively low, but progressively increasing, levels (), conferring follicular sensitivity to FSH.This is the proliferative signal for granulosa cells, which start to replicate and synthesize steroid hormones, inducing antrum formation and supporting oocyte maturation.At the early follicular phase, relatively low LH levels are produced, and it has been commonly accepted that these are enough to mediate androstenedione synthesis by LHCGR-expressing theca cells ().In contrast, relatively high levels of FSHR are exposed at the surface of granulosa cells, which also express low levels of LHCGR (), suggesting a physiological role for the putative interaction reported to occur between these two receptors () in modulating gonadotropin signals ().If the coexistence of FSHR and LHCGR has biological importance, it is exquisitely granulosa cell-specific, because FSHR is absent in theca cells.Most importantly, these data suggest that LH-and FSH-dependent synergetic action in granulosa cells, not provided by each gonadotropin per se, is required to properly guide follicular growth.At this stage, the best-known role of theca cells is still to supply androstenedione, as the substrate for estrogen production in granulosa cells, supporting follicular growth and oocyte maturation/ metabolism.As pituitary FSH release and follicular FSHR expression decline, LH levels increase, along with progression toward the large antral stage, when follicular growth is gonadotropin and estrogen dependent.However, both FSH and LH production still coexist at this stage, with both proliferative and apoptotic signals occurring in the dominant and atretic follicles, respectively.In the late follicular phase, LHCGRs fully replace FSHRs in the granulosa cells of the dominant follicle to induce both ovulation and the changes in metabolic state necessary for luteinization (i.e., massive, exclusively LH-driven progesterone production).Therefore, although both FSHR-and LHCGR-mediated life and death signals are simultaneously present in granulosa cells, only LH-dependent signals of increasing potency are delivered to theca cells throughout the entire follicular phase up to ovulation.Thereafter, the second half of the menstrual cycle depends on progestational signals transitorily supported by LH in the corpus luteum.
Only in the case of pregnancy is corpus luteum function maintained by trophoblast hCG, because the LH levels decrease as a consequence of the negative feedback exerted at the hypothalamic-pituitary level by high progesterone concentrations.Therefore, physiologically, the steroidogenic LH activity in the ovary is not naturally replaced by hCG before this stage.The progression of pregnancy features steadily increasing progesterone levels, which, during the first  to  weeks of gestation, result from the hCG action on the corpus luteum ().It is well known that hCG stimulates the maternal androgen production required for fetal development and pregnancy progression ().Moreover, it seems that both placental and adrenal androstenedione and testosterone play a role in cervical and myometrium remodeling and parturition (), and excessively high androgen levels might compromise maternal health ().Apart from these functions, little is known about the direct action of hCG on androgen synthesis, although the role of the gonadotropin has been classically associated with progesterone synthesis during pregnancy.hCG is secreted in high amounts, especially in the first trimester of pregnancy, and acts as an essential and potent steroidogenic factor.Other functions have been, however, hypothesized for hCG, including immunosuppressive and angiogenic functions, especially during the early weeks of pregnancy (), and the capability of enhancing steroid-mediated signals by activating cAMP-and extracellular-regulated kinase / (ERK/)-mediated production of the progesterone receptor in endometrial cells ().The exposure to maternal hCG is crucial for fetal sex steroid production and activation of the hypothalamicpituitary-gonadal (HPG) axis, which affects fertility potential in adulthood ().After birth, maternal estrogens decline in the newborn, leading to the increase of FSH and LH, characterizing the neonatal period termed minipuberty.This results in a surge of pituitary gonadotropins of a magnitude only comparable to the levels obtained much later during puberty ().
FSH and LH signaling are also fundamental for male gametogenesis, because these hormones act on Sertoli and Leydig cells, respectively, providing mechanical and endocrine support for sperm production.The hormonal control of spermatogenesis and, especially, its dependence on FSH or LH, are extremely species-specific among mammals (), although testosterone is, in general, an essential requirement for the progression of gamete maturation.Testosterone is produced by Leydig cells on LH stimulation and sustains Sertoli cell function and spermatogenesis progression, albeit being partially converted to -b-estradiol by the aromatase enzyme, promoting antiapoptotic signals, likely together with gonadotropins (, ).Given the steroidogenic role of Leydig cells, which express LHCGR, but not FSHR, it is understandable that hCG found clinical utility in replacing LH functions.Because the use of LH for treatment of male HH is still limited, even in the era of recombinant gonadotropins, no substantial data are available to differentiate its action from that of hCG in males.
Studies that focused on the metabolic fate of gonadotropins indicated that only ~% of hCG is excreted in the urine, and the retained hormone is resorbed and degraded mainly in the kidney and, to a lesser extent, the liver and ovary ().In the kidney, these molecules are metabolized to b-core fragments deprived of galactose, sialic acid, and CTP fragment, suggesting that these modifications are required for urinary excretion.Sugar moieties play a key role in establishing the circulatory half-life of LH and hCG.The routes and rates of LH/CG distribution and elimination were compared in rats and piglets, revealing that a high quantity of radiolabeled porcine LH is accumulated in the kidneys within  minutes of injection, the equine chorionic gonadotropin (eCG) plasma concentration is % after  hour, and the hormone is not accumulated in any organ ().Taking together, these data indicate that LH is eliminated from serum by renal trapping, resulting in rapid removal compared with CG.Other, minor routes for gonadotropin elimination can be found in the liver, through binding of sulfated oligosaccharides to a specific receptor (SGGnM) expressed in the Kupffer cells ().Gonadotropins are subsequently processed in the kidney to be excreted with urine as residual, highly similar LHb and hCGb core structures, identical to the original pituitary and trophoblast core molecules ().This is due to the relatively high stability of the gonadotropin structure, which is nearly identical among LH and CG molecules of humans and other primates (), suggesting that urinary excretion of highly similar gonadotropin core metabolites might be evolutionarily conserved.
In summary, LH and hCG are involved in the regulation of multiple physiological functions, but their specificity is underrated due to their action through the same receptor and clinical experience derived from the use of readily available hCG only in the treatment of HH and in controlled ovarian stimulation (COS) for ART.In the latter case, gametogenesis could be clinically supported by administration of exogenous FSH and hCG, thereby at least partially replacing LH action.However, clinical data have not provided representative models for understanding LH-and hCG-specific functions in vivo to date.For instance, stimulation of multiple oocyte production in the clinical setting of COS is far from replicating the natural oocyte selection, because it results in multifollicular development in a mono-ovulatory species.This effect is due to the pharmacological gonadotropin dosages, which do not necessarily elicit physiological patterns of estrogen production and oocyte selection.

Evolutionary convergence: trophoblast LH and pituitary CGs
The endocrine adaptation to pregnancy results in different, interesting evolutionary strategies exhibited across the phylogenic tree of mammals.The analysis of the lhb genomic locus of several species suggests that the CTP fragment might be produced by a number of organisms by frameshift of the gene transcription ().Data suggest that the concept relating the CTP fragment with placentation of primates could be extended to all mammals () that share the potential to produce glycoproteins bearing the CTP peptide, which likely possesses the key characteristics of hCG.A proof of concept might be provided by the bovine, which produces a pregnancy LHb variant featuring a CTP fragment.This molecule is produced by decryption of the 9 region of the lhb gene, resulting in a glycoprotein hormone that is, however, poorly O-glycosylated and displays a shorter half-life compared with hCG, not supporting the evolution of CG molecules in bovines ().
A placental gonadotropin was described in equids long ago (, ).This hormone is known as eCG, suggesting a similarity to the CG molecules of primates.However, both b subunits of equid pituitary LH (eLH) and trophoblast eCG are products of the same lhb gene.They differ in their source of production and N-linked glycosylation, which is greater for eCG than for eLH, with O-linked glycosylation crucial for both hormones to maintain binding activity ().Moreover, both eCG and eLH have demonstrated binding capability for fshr (), with results even greater for eLH than for eCG (), suggesting a role in mediating FSH-like signals.The eCG capability of promiscuous fshr activation was described in most mammals (, ), although it was negligible in horses ().These gonadotropins consist of glycosylation variants, providing a case for evolutionary convergence between equids and primates, with different strategies adopted to support the same physiological process.
Interesting data were provided by studies of primates, which seem to be an evolutionary counterpart to bovines and equids when considering LH and CG.In , LH bioactivity in the New World marmoset monkey Callithrix jacchus was demonstrated to be produced by a pituitary monkey CG sharing ~% identity with hCG ().Monkey CG displays multiple activities regulating development, gametogenesis, and pregnancy, likely owing to a glycosylation pattern similar to human LH and a CTP structure similar to hCG.In contrast, the marmoset monkey LHCGR lacks the amino acid sequence encoded by exon  of the gene, which corresponds to an extracellular portion of the receptor ().Although the molecular structures of gonadotropin and their receptors have been detailed in the next section ("Different Sources, Molecular Structures, and Biochemical Properties"), the presence of a CG molecule and the lack of an exon -encoded sequence in C. jacchus LHCGR (also known as LHCGR type II) in the entire New World monkey lineage provide interesting information about LH/CG receptor functioning, which reaches its maximum complexity in primates ().A previous study suggested that the CTP fragment is essential to induce LHCGR type II activation ().One study described an interesting case of an -year-old male patient with Leydig cell hypoplasia characterized by the absence of exon -encoded portion of the LHCGR, who was unresponsive to endogenous LH ().Thus, this patient had relatively high serum LH and very low testosterone levels, delayed pubertal development, and small testes, indicating a deficit in the LH signal.However, testosterone biosynthesis and spermatogenesis were recovered with hCG treatment.hCG also induced cAMP production during a functional analysis in vitro assessed in exon -deficient LHCGR-transfected cells, which LH failed to stimulate despite binding to the receptor ().This clinical case confirmed the importance of the amino acid region encoded by the exon  of LHCGR to discriminate between the two natural ligands, supporting the concept of coevolution of the ligand-receptor structure as a strategy to regulate gametogenesis and placentation in primates (Fig. ).
Although it might be intuitive to find the structure-function relationship of placental gonadotropins, the role of hCG and hCGb molecules found in the human pituitary () is still unknown.They were detected in the serum of both men () and women () and would be released in a pulsatile fashion.It was postulated that pituitary hCG molecules might play a role in the regulation of the menstrual cycle () and ovarian pathogenesis (); however, further evidence is needed to support this issue.

Different Sources, Molecular Structures, and Biochemical Properties
The genetic differences of LH and hCG reflect gonadotropin-specific molecular structures, posttranslational modifications, and biochemical properties at least partially established in the secretory pathway involving the endoplasmic reticulum and Golgi apparatus of the source cell ().The secretion of LH by gonadotrope cells of the anterior pituitary is controlled by the GnRH (), a peptide produced by the hypothalamus, under kisspeptin regulation (), in a pulsatile fashion and released into the portal bloodstream.GnRH binds its seven-transmembrane "A variety of LHb molecules can be produced in humans." receptor expressed in gonadotrope cells, mainly activating phospholipase C (PLC)-, ERK/-, b-catenin-, calmodulin-, and PKA-dependent signaling (, ).The preferential LH or FSH synthesis depends on the frequency of GnRH pulses.Low frequencies are linked to FSH production, and higher frequencies are synchronized with waves of LH synthesis ().In women, the preovulatory stage of the menstrual cycle is characterized by a GnRH surge corresponding to the LH increase, inducing ovulation.Given the dependence of gonadotropin production on kisspeptin and GnRH, a new model of ovarian physiology was proposed, in which follicle maturation and selection, ovulation, and luteal phase occur under the strict control of the neuroendocrine system ().The relationship between gonadal and pituitary functions encoded by a single Lhb gene.Mouse Lhb acts through its receptor, which displays the exon 10-encoded portion at the hinge region, and regulates both gametogenesis and pregnancy.A CTP fragment appears as a product deriving from the equid Lhb gene, which is the source of a placental choriogonadotropin.Although pituitary eLH regulates gametogenesis, eCG is specific for pregnancy in equids.However, they act on a common receptor with the exon 10-encoded sequence.The New World monkey Callithrix jacchus has a peculiar LH/CG system.Both gamete maturation and pregnancy are supported by a unique CGB gene encoding transcripts of both pituitary and placental origin and displaying the CTP fragment.Physiological functions are ensured by the LH receptor type II, lacking the exon 10-encoded portion, which binds the CGb molecule in replacement of the missing LH.In humans, LH and hCG isoforms are encoded by specific genes belonging to a genetic cluster.However, pituitary LH lacking a CTP and placental hCG possessing a CTP appear as major products, specifically regulating gametogenesis and pregnancy, respectively.Both human LH and hCG act through the same receptor displaying the amino acid sequence encoded by exon 10, which is fundamental for activating hormone-specific intracellular signaling.Deletion of LHCGR exon 10-encoded region leads to a truncated receptor capable of binding both LH and hCG but resulting in impaired LH signaling and male infertility.Production of CGa, encoded by CGA genes, is required for proper dimer formation with b subunits, although CGas were not included in the image.
was shown by experiments in Lhr knockout and ovariectomized mice ().In both of these models, high expression of gonadotropin subunit genes occurred, reflecting morphological changes in gonadotrope cells, which display secretory granules larger than those in wild-type mice.In contrast, in GnRHdeficient mice expressing low gonadotropin mRNA and protein levels, gonadotrope cells were smaller and featured fewer secretory granules.A variety of LHb molecules can be produced in humans, although these have not yet been completely functionally characterized.They differ in glycosylation patterns, resulting in specific molecular weights ().
The secretion of hCG by trophoblast cells occurs in a nonpulsatile, increasing manner, reaching the peak around the first trimester of pregnancy, and is not coupled to GnRH production.A wide variety of hCG isoforms and glycosylation variants are produced during this period, promoting trophoblast invasion of maternal decidua.Most interestingly, and in contrast to LH, hCG production is not subjected to immediate downregulation by steroid hormones.It could be speculated that steroidogenic hCG-mediated signals are constantly delivered during pregnancy as a requirement to maintain proper progesterone production, although the different nature of LH is aimed at more transitory events, such as luteinization, which need not be prolonged.A suggestive explanation of the molecular mechanism underlying constant hCG activity could be provided by the discovery of an ~-kDa truncated form of LHCGR, specifically expressed by placenta and choriocarcinoma cells, the presence of which is concomitant with the absence of hCG downregulation ().The presence of this truncated receptor would be opposed to the appraisal of full-length (~-kDa) LHCGR at the term placenta, which suggests the existence of a feedback mechanism regulating hCG action via receptor downregulation.Although these findings should be independently confirmed, it is known that hCG production is constant but undergoes qualitative dynamic changes throughout pregnancy.It has been suggested that the pattern and abundance of hCG molecules is individually specific; thus, choriogonadotropin acts as a dynamic, autocrine factor that changes qualitatively throughout the first trimester of pregnancy ().hCG isoforms consist of different polypeptide products selectively transcribed by CGB genes (), and the oligosaccharide structures differentially linked to the hCG backbone determine the glycosylation variants and depend on the enzymatic milieu of trophoblast cells, which differs from that of pituitary gonadotrope cells ().
The effects of oligosaccharide structures, especially O-linked, are apparent in the serum half-life differences between the two hormones, which is  minutes for LH and  hours for hCG ().In particular, Olinked glycosylation is abundant in the CTP fragment of hCG and could be important in determining hormone-specific physiological roles, providing a strategy for developing chimeric gonadotropins with slow metabolic clearance ().In gonadotrope cells, gonadotropin glycosylation is modulated by a number of hypothalamic and gonadal endocrine factors, such as estrogens and androgens ().These steroids could also affect sialylation and sulfation of the oligosaccharide, further modulating the half-life and biopotency, thus extending the qualitative range of signals delivered to the gonads.In contrast, sulfation of oligosaccharides is critical for hormone half-life and bioactivity.This posttranslational modification is evolutionarily preserved among glycoprotein a subunits, from teleost fishes to mammals ().Finally, it is reasonable, albeit speculative, that the pulsatile release of a short half-lived LH would provide a finetuned stimulus optimized to regulate life signals mediating follicle growth and that hCG molecules would trigger the relatively potent stimulus for sustained and prolonged progesterone synthesis and angiogenesis.In summary, two different gonadotropins, LH and hCG, should be recognized at the receptor level to reach a finer level of regulation, not allowed by a unique ligand, mediating specific intracellular signals required to optimize reproduction and development.

Production of a and b subunits
The production of glycoprotein a subunits is a ratelimiting step for gonadotropin heterodimer formation.The a subunit, encoded by the .-kb CGA gene (), transcripts of which are found in pituitary and placenta, allows for glycoprotein hormone heterodimer assembly occurring in the endoplasmic reticulum of source cell ().Both hCGa and b subunits are characterized by three loops defined by a cysteine knot, and experimental evidence has proved the importance of the second loop of the a subunit in dimer formation.These results were obtained using chimeric molecules, in which the second loops of the hCGa and b subunits were swapped, resulting in b-b homodimers capable of receptor binding and activation of signal transduction, albeit at lower levels than in wildtype hCG, and inactive a-a homodimers ().The knowledge of how the formation of glycoprotein hormone heterodimers occurs was improved by several attempts to develop the crystal structure of hCG during a -year period from  to  (-).These models revealed interesting features of the molecule, mostly the seat belt-like structure composed of a segment of the b subunit wrapping around the a subunit loop aL linked by a disulfide bridge between two cysteine residues at positions  and  of the polypeptide chain.However, a complete view of the tridimensional dimer structure was strongly hampered by the oligosaccharide structures bound to hCG.Again, experiments using mutant a subunits lacking the disulfide bonds between cysteine residues at positions - and - provided a further step in understanding the procedure of gonadotropin heterodimer formation ().Disruption of these structures did not substantially affect hCG or FSH heterodimer formation, although it negatively affected LH heterodimerization in transfected Chinese hamster ovarian (CHO) cells and in the rat pituitary tumor cell line, GH.In contrast, N-linked glycans at positions  and  are required for efficient hCGb folding and formation of disulfide bonds between the residues at positions -, -, and - ().These data suggest that regions recognized by the a subunit for assembly are different in LHb and hCGb.
New insights on the formation of LH and hCG dimers were provided by recent advancements allowing for the evaluation of molecular interactions using a bioinformatic approach.This in silico analysis revealed that dimer assembly might follow different b-subunit-specific modes, depending on the presence or absence of the hydrophobic tail of LHb and hCGb, respectively ().The LHb subunit is not completely folded when docking with the a subunit occurs.In such cases, the a subunit acts as a scaffold, using the cystine knot to enhance b-subunit cystine knot formation.The heterodimer is then stabilized by the interaction between loop  of the a subunit and the b-subunit hydrophobic tail, which form the seat belt-like structure.This LHspecific mode of dimer formation was named "wraparound" and differs from the assembly of the hCGa-b dimer, which was named "threading," to explain the mechanism by which the a subunit passes between a b subunit folded before the docking.An alternative mechanism, similar to the "wraparound" model, was also proposed for hCGa-b dimer formation (, ).However, these data suggest that a different heterodimer assembly might have arisen as a strategy to control the production of the two hormones.Although hCG assembly is efficiently performed, in vitro experiments using chimeric gonadotropins revealed that LH assembly and secretion are less vigorous ().The faster rate of hCG secretion compared with LH secretion presumably reflects their massively constitutive and GnRH-regulated secretory pathways, respectively.The CTP fragment of hCG plays a role in determining heterodimer formation, because CTP-truncated forms of the chorionic hormone resulted in a % decreased efficiency of dimerization.Removal of the LHb C-terminal octapeptide increased the rate of hormone secretion by transfected CHO cells, confirming the relevance of the C-terminal region of LH and hCG for their physiology.
Previous attempts to produce recombinant LHb and hCGb in transfected GH cells in vitro revealed the role of the N-terminal region in the disulfidelinked aggregation of LHb subunits.In contrast, hCGb was exclusively secreted as a monomeric molecule.Further investigation using mutant gonadotropins demonstrated that N-glycosylation on asparagine at position , which LHb lacks, prevents hCGb aggregation in culture medium ().These data indicate that glycosylation in the N-terminal region of the two gonadotropins plays a role in the maintenance of the correct folding and secretion in the absence of the a subunit (Fig. ).This finding might explain the measurable presence of free hCGb subunits during pregnancy, when high activity of CGB genes leads to massive protein production and free LHb subunits are relatively rare ().

LHb subunit
LHB gene transcription is stimulated on GnRH binding to its receptor, activating Gq/ proteins and stimulating phospholipase C to mediate inositol ,,-triphosphate and diacylglycerol pathways.These intracellular messengers lead to protein kinase C activation, intracellular calcium increase, and b-catenin signaling regulating the expression of the LHB gene.The latter is under the control of GATA and chicken ovalbumin upstream promoter-transcription factors (, ), and the secretion of LH is regulated by the increase in intracellular calcium (-).The LHb monomer was described in the s ().It is an ~-kDa glycoprotein of  amino acids subjected to posttranslational modifications, which provide .variants of the hormone, with molecular weights spanning from  to  and featuring specific bioactivity ().The core fragment is structurally close to that of hCGb, except for the absence of the CTP fragment.The nomenclature of the amino acid position within LHb and hCGb subunits originated from these first sequence determinations (-), which excluded the -amino acid-long signal peptide.A new nomenclature that included the length of the signal peptide was proposed later () and was used to indicate the amino acid positions in the present report.
Glycosylation, sulfonation, and sialylation are the main LH modifications, which occur at different rates during the ovarian cycle () and with age ().In particular, the LHb subunit classically displays one Nglycosylation site at position  of the polypeptide chain, and two additional oligosaccharide structures are linked to the a subunit ().LH heterogeneity consists of a prevalent glycoform carrying three oligosaccharide structures, except during midcycle when diglycosylated LH is the main LH variant produced.Moreover, the acidic forms of LH molecules increase with age and are mostly present in elderly women.Because the removal of these oligosaccharide structures by endoglycosidase treatment did not substantially change the LH steroidogenic activity, their functional importance might be related to other aspects of physiology ().Oligosaccharides can be sulfonated or sialylated, resulting in different half-lives of the glycoprotein.Sulfated oligosaccharides consist of branches terminating in SO-GalNAc b,, and sialylated oligosaccharides consist of a number of different structures featuring two or three branches and one to three sialic acid moieties ().LH molecules with two or three sulfonated N-acetylgalactosamine [SO()-GalNAc] residues have shorter half-lives than less-sulfonated LH, suggesting their rapid removal by hepatic Kupffer cells.In contrast, higher sialylated gonadotropin isoforms have extended half-lives, likely owing to the masking of the sulfonated oligosaccharides to SGGnM binding and sequestration (, ).However, rather than SO(), sialic acid might be linked to LH GalNAc residues, likely resulting in an extended half-life ().

hCGb isoforms and glycosylation variants
In humans, a number of CGb isoforms and variants are provided by transcription of CGB genes and different patterns of glycosylation, respectively.These types of hCG are known as "classical" hCG, hyper-and hypoglycosylated hCG, nicked isoforms and hCG lacking the CTP fragment, core fragments, and free b subunits.These molecules represent a palette of multiple hCGbs differentially detectable as urinary products by specific immunoassays (, ).
Although in-depth functional characterization of CGb isoforms in vitro is missing, the expression pattern of CGB genes might be a determinant of the status of pregnancy or miscarriage.Although the regulation of CGB gene transcription is unclear, it is likely under the control of growth factors, cytokines, ligands of the nuclear peroxisome proliferatoractivated receptor-g, and steroid hormones, acting through activation of cAMP-mediated signals ().In normal pregnancies, all CGB transcripts were found, especially CGB, CGB, CGB, and CGB, which achieved to ,-fold greater expression levels than CGB and CGB genes ().An ectopic pregnancy is characterized by perturbation of CGB expression patterns, featuring a relatively high amount of CGB and CGB transcripts.In contrast, globally reduced CGB expression has been associated with miscarriage.Relatively high amounts of CGB and CGB gene transcripts were found in the testes of healthy males, suggesting that they might play a still unknown role in male reproduction ().
Glycosylation is the posttranslational modification providing .hCGb variants, depending on the combination of glycans specifically elaborated by the source cell and attaching two potential N-and four potential O-glycosylation sites in hCGb ().hCGb isoforms possessing only one N-glycan are known and only three of four O-glycosylation sites are typically decorated with carbohydrate (, ).Moreover, # glycoforms of the a subunit were found, differing by sialylation, oxidation, and N-terminal truncation ().Because hCG and LH are administered for infertility treatment as a mixture of different glycoforms resulting from the manufacturing processes and potentially featuring specific biochemical properties and bioactivities, the production of homogeneous hormone samples by chemical synthesis should be of great interest for clinical applications ().During pregnancy of aneuploid fetuses, the profile of hCG glycoforms is different from that detectable in pregnancy of normal karyotyped fetuses ().Although fully informative characterization of these hCG glycoforms in vitro is lacking, it was suggested that they are related to specific biological activities and functions, mainly angiogenic (), in vivo ().These conclusions were driven by experiments evaluating cell growth and migration in vitro and mediated by hyperglycosylated hCG molecules (hCG-H) (), predominantly produced during the early stages of pregnancy by extravillous cytotrophoblasts or choriocarcinoma, and consistent with the positive regulation of early trophoblast invasion ().hCG-H would have lower steroidogenic and greater proliferative potential than the "classical" form of hCG produced later ().It should, therefore, be secreted during the very early days of pregnancy as an essential mediator of cell proliferation and maternal tissue invasion by fetal cells.Thus, insufficient hCG-H during the first days of pregnancy might be predictive of miscarriage (), highlighting the fundamental role of this molecule for placentation and embryo development.Similar conclusions were drawn by analyzing the sera of women experiencing recurrent miscarriages, in which antitrophoblast antibodies inhibited the hCG-H release by the JEG- cell line in vitro ().Alternative, "hyperbranched" glycoforms linked to both a and b subunits are predominantly produced during aberrant pregnancy and by choriocarcinoma rather than in normal pregnancy, suggesting that the activity of Golgi-processing enzymes occurs differently in malignancy ().Hyperbranching might reflect the presence of free a and b subunits, by which dimer formation is impaired by largely tri-and tetra-antennary glycans, in contrast to those biantennary glycans associated with classical hCG ().The angiogenetic potential of hyperglycosylated hCGs is a crucial issue for pregnancy success, because proper blood flow is required to avoid embryo hypoxia.This role might be exerted via activation of cAMP-responsive elements located in the promoter region of vascular endothelial growth factor-encoding genes ().It has been proposed that angiogenic functions and trophoblast invasion of hCG-H could rely on growth factor-like activities putatively mediated through interaction between the hormone and TGFb receptor II, independently of classical hCG signaling () (Table ).A similar mechanism of action was suggested to explain the tumorigenicity of free hCGb subunits in BRCA genedefective breast cancer ().This is a suggestive hypothesis requiring further confirmation before being accepted, because an independent study revealed the opposite findings and suggested that experimental biases, such as TGFb contamination in the hCG preparations, might have affected the results ().Although the molecular mechanism underlying the proliferative potential of hCG-H is still unclear, it has been confirmed that these molecules are secreted by several tumor cells, even in the male (), likely as a glycosylated product occurring after metabolic reprogramming of tumor cells ().These data suggest that hCG-H might be a tumor marker and promoter ().
LH and hCG binding to the human LHCGR: binding differences compared with nonhuman receptors Human gonadotropins display binding capability to nonhuman receptors.Although indicative of the overall structure preservation of these ligands and receptors during evolution, this is also relevant for clinical treatment of human infertility.LH and hCG dosages for clinical purpose are established by evaluating their biological activity against a standard using animal models not expressing the human receptor (), suggesting some limits to their translation to humans and usage in clinical practice.The first evidence of LH and hCG receptor binding was provided several decades earlier, by in vitro studies using rat Leydig cells.These experiments found similar sets of binding sites and binding capacity for both hormones to the rat Lhr, evaluated by equilibrium association constants (Ka; hCG Ka = . 3  2 M; LH Ka = . 3  2 M) and association rate constants (K  ; hCG K  = . 3   M/min; LH K  . 3   M/min) ().However, greater binding affinity was suggested for hCG compared with LH, owing to its longer persistence at the receptor level and greater halftime of the bound hormone (hCG, . hours; LH, . hours), resulting in a reduced dissociation rate for hCG.These results suggest that the two hormones interact differently with the receptor, as a consequence of distinct amino acid sequences.It was observed that the maximal hCG-mediated cAMP increase occurred at relatively low (,%) Lhr occupancy in rat Leydig cells, adumbrating the existence of "spare" Lhr ().This concept arose by evaluating the discrepancy between hCG binding and dose-response curves calculated for cAMP, which appeared to be left-shifted.It has been recently used to explain the putative, steroidogenic activity exerted by LH during the antral phase of folliculogenesis (, ).
The putative existence of "spare" LHCGRs in the human ovary can be explained by the requirement for sustained androgen synthesis by theca cells, avoiding receptor downregulation by LH.If so, hCG must have a different action at the receptor level during pregnancy, when the hormone is massively produced and constantly stimulates progesterone synthesis, somehow avoiding LHCGR downregulation.Although suggestive, these are largely conjectures, because the existence of "spare" LHCGRs was only suggested in vitro.Because hCG is not the natural ligand of rat Lhr, the parallelism between in vitro findings using rat Leydig cells and the physiology of the human ovary should be interpreted cautiously ().In contrast, LH-like signals might be driven by FSH through FSHR-LHCGR heterodimerization in granulosa cells (), which should be favored by the ~: ratio between the LHCGR and FSHR amounts at the early antral stage ().Moreover, ligand affinity to rat and human receptors might not be similar, as suggested by hCG and bovine LH, which both exhibit similar affinities for the rat Lhr but different binding affinities for LHCGR (-to ,-fold better for hCG than for bovine LH) ().These differences are due to an isoleucine residue falling within the C-terminal end of LRR of LHCGR, comparable to a serine residue in LRR of the rat Lhr, which would determine the LH-specific binding affinity ().In vitro comparisons of human LH, hCG, and some of their nonhuman, rat, equine, bovine, ovine, and porcine counterparts in the mouse tumor Leydig MLTC cell line, revealed molecule-specific control of adenylate cyclase activity, raising the question of whether hCG could be a reliable reference ligand in nonhuman LH receptorexpressing systems ().These data suggest that Lhr and LHCGR might not be comparable and might mediate receptor-specific responses.Although the assumption of "spare" ovarian LHCGRs should at least be confirmed by binding experiments using radiolabeled LH and cells expressing the human LHCGR, it is clear that most of our knowledge of LH and hCG binding has been provided by experiments using cell models expressing nonhuman receptors, relying on the intra-and interspecies promiscuity between gonadotropins and their receptors but ignoring their differences.Conformational changes of LH and hCG occurring on receptor binding were first demonstrated using rat gonadal cells ().The importance of specific contact sites, such as the intercysteine loop sequence of both LHb and hCGb, was also demonstrated, revealing that the amino acid region - of these subunits is exposed on the surface of the molecule and participates in rat Lhr binding (), along with lysine residues at position  of hCGb and position  of both gonadotropins ().However, the hCGa subunit directly interacts with the LHCGR extracellular domain (), participating in hormone receptor binding, and it cannot be excluded that a similar interaction occurs with LH binding.
Informative results regarding LH and hCG binding affinity for LHCGR were provided by experiments evaluating the displacement of radiolabeled hCG by increasing the molar concentrations of the hormones incubated together with membrane lysates of LHCGRtransfected COS- cells ().LH displayed an approximately eightfold greater, albeit not statistically different significantly, half-maximal inhibitory concentration compared with hCG (IC; hCG IC = .pM; LH IC = .pM), demonstrating a quite similar binding affinity for LHCGR between the two gonadotropins.In any case, these experiments should be repeated, evaluating the displacement of radiolabeled LH to draw definitive and clear-cut conclusions about the binding features of both molecules.In contrast, most of the current knowledge depicting the interaction between LHCGR and its ligands was provided by classical experiments using mutated human receptors or chimeric hCG.It is known that hormone b subunits contact specific amino acid residues of LHCGR LRR b strands, especially  and  (), which play a role in the formation of a "sled-like" tridimensional structure typical of the gonadotropinreceptor extracellular domain and in hormone binding and activity ().Analysis of the chimeric hCG b-b dimer confirmed the need for this subunit for receptor binding.However, this dimer bound two receptor molecules with threefold lower affinity than classical hCG and failed to elicit any cAMP response ().These results have demonstrated that the a subunit is involved in LHCGR binding and activity.Experiments using an hCG analog, obtained by fusing the C-terminus of the a subunit and the N-terminus of the b subunit through a CTP fragment, confirmed that both the N-and the C-terminal portions are involved in receptor binding and activation (), and the seatbeltlike structure of hCG is only minimally involved in LHCGR binding ().Finally, both the a and b subunits of hCG possess the first three b-hairpin loops, structurally similar to those of the TNF and the nerve growth factor, not involved in receptor binding ().It is remarkable that the corresponding structures of TNF and nerve growth factor were instead crucial for interacting with their respective receptors.
Interesting results were provided by mutagenesis of LHb and hCGb, revealing hormone-specific biochemical features embedded in the polypeptide structure of the two molecules.The glutamine residue at position  of the b subunit plays a key role in dimer formation.Substitution of this glutamine with a basic amino acid residue, arginine or lysine, resulted in subunit association decreasing to ,% compared with the wild-type and formation of inactive, mutated LH and hCG dimers, which failed to induce progesterone synthesis in the MA- cell line.Neutral (alanine) or acidic (glutamic acid) residue substitution at position  resulted in mildly (% to %) decreased subunit association and a lack of mutant LH binding, although mutant hCG retained full activity ().Although the amino acid residue at position  is crucial for both LH and hCG heterodimer formation, structural characteristics intrinsic to the protein chains result in functional differentiation between LH and hCG despite the similar sequences.Most importantly, these data suggest that the two gonadotropins might bind the receptor differently; however, further mutagenesis experiments are needed to fully clarify this issue.Overall, they seem to interact similarly with the LRR domain (), and a second, lessknown, binding site falling within position - and belonging to the hinge region of LHCGR might be involved in hormone-specific contacts.Advancements were provided using a bioinformatic approach evaluating the LH-and hCG-specific interaction with the hinge region.In this case, an extroflexion consisting of a sulfated tyrosine located at position  (sTyr) would play a key role in discriminating between the two hormones, relying on a specific spatial conformation of the receptor hinge region (Fig. ).The protein segment carrying the sTyr features a "Ushaped" structure displaying proximity between the amino acid sequence encoded by the LHCGR exon  and an adjacent helix.Both LHb and hCGb first bind the LRR domain of the receptor.However, although the spatial occupancy provided by hCG binding to LRRs contributes to the LHCGR conformational change by contacting the whole "U-shaped" structure, the smaller size LH needs to interact with the sTyr extroflexion to induce proper conformational assembly of the receptor ().The deletion of the amino acid sequence encoded by exon  would result in a modification of the "U-shaped" structure, consisting of the shift of the adjacent helix, which spatially replaces the exon -encoded sequence, shifting Tyr to a special position not permissive for LH accommodation.Thus, LH signaling would be impaired () and hCG would retain its functional properties owing to preservation of the "U-shaped" structure binding and conformational change of LHCGR.These data were supported by experiments altering the exon -encoded sequence by introducing a double proline mutation at positions  and .In this case, disruption of the "U-shaped" structure with preservation of the proper spatial location of the sTyr residue would negatively affect hCG but not LH signaling ().
Because LH and hCG display a specific interaction with LHCGR, different conformational changes of the receptor can occur, depending on the hormone.In the HEK cell line, coexpression of signaling-deficient LHCGR and binding-deficient LHCGR () allowed for evaluation of the ligand-induced intermolecular cooperation ().Although treatment by hCG was linked to full cAMP activation, LH failed to induce an intracellular increase in second messenger ().An hCG-induced cAMP increase was the result of binding-deficient receptor activation by signalingdeficient LHCGR capable of hormone binding, which relies on receptor dimer formation and the ligand-receptor complex undergoing specific conformational changes.This is the so-called LHCGR transactivation, and it was suggested to occur during hCG treatment, with LH able to induce mainly self cisactivation of receptors capable of hormone binding ().These results and, in general, receptor cis-and transactivation were independently supported (, , ), even if the opposite results were also reported (), questioning the concept of ligandspecific functional rescue between LHCGR molecules ().
In summary, as a result of common evolution, cross-interaction between ligands (LH and hCG vs murine lh) and receptors (human LHCGR, murine lhr) from different species can be demonstrated in vivo and in vitro, together with species-specific patterns of ligands for one receptor as adaptations to maintain effective biological responses.However, human LH and hCG display their own specific molecular interactions with human and nonhuman receptors, resulting in hormone-dependent modulation of downstream intracellular signaling and physiology, which is considered in detail in the next paragraphs.

LH-and hCG-Specific Intracellular Events
LHCGR binding to its ligands triggers a number of subsequent events mediating the activation of multiple signal transduction pathways ().These events start after hormone interaction with its high-affinity binding site located in the extracellular domain of the receptor, which, however, is not capable of generating intracellular signaling per se.The bound receptor undergoes a conformational change affecting the hinge region and, subsequently, the transmembrane domain.However, contacts between the extracellular domain and loops (), especially the second and third extracellular loops, are necessary for proper signaling activation.These secondary, lowaffinity contacts established by the hormone compelled to interact with extracellular loops and the hinge region play a key role in signal generation ().The spatial conformation of the activated LHCGR is linked to different, independently activated, signaling cascades, depending on various cell-specific intracellular interactors of the receptor, which mainly consist of G proteins () and b-arrestins ().Although the presence of LHCGR at the cell surface is linked to weak basal signals existing as an equilibrium between stimulatory and inhibitory signals, maximal production of high-affinity signal occurs on hormone binding.
Although the intracellular events described are overall common to all glycoprotein hormone receptors, the existence of ligand-LHCGR-specific interactions suggests that qualitatively and quantitatively different patterns of intracellular signaling cascades might be differentially activated by LH and hCG.These features could rely on peculiar LHCGR conformational changes induced by ligands and the presence of other interacting glycoprotein hormone receptors and the intracellular enzymatic milieu of target cells.All these factors likely contribute jointly to differentiating LH and hCG physiology.

Classical views and insights on LHCGR-mediated signaling
Knowledge of LHCGR-mediated intracellular events has progressively increased during the past few decades, revealing a complex picture of gonadotropin functions not explainable exclusively by the old concept of steroidogenesis as the main endpoint of both LH and hCG function, exerted via cAMP/PKA activation and intracellular calcium ion (Ca + ) increase.This classical assumption presumably originated when cAMP and steroid hormones were the main-or even only-molecules analyzable using the first assays available.Modern experimental techniques have revealed the existence of several intracellular LHCGR interactions and multiple signaling cascades, requiring a re-evaluation of LH-and hCG-mediated signals.
It is common knowledge that LH and hCG induce simultaneous increases of the second messenger cAMP and Ca + through the LHCGR ().These two events occur relatively early after receptor activation, within , minute (, ), and belong to two separate, G-protein-dependent signaling pathways ().Spatial conformation of the activated receptor leads to G-protein stimulatory Gas subunit dissociation from the bg dimer, thus activating the adenylyl cyclase membrane enzyme, which, in turn, catalyzes the conversion of ATP into cAMP.This second messenger, before its metabolization to AMP by phosphodiesterase enzymes, induces PKA activation and transcription factor cAMP response element-binding protein (CREB) phosphorylation ().However, relatively high intracellular cAMP concentrations were linked to proapoptotic effects in granulosa cells, along with progesterone synthesis and androgen conversion to estrogens (, ).In the theca cell, phosphorylated CREB (pCREB) binds CRE DNA target sequences, modulating the transcription of steroidogenic enzyme-encoding genes, such as STARD and CYPA, and synthesis of androstenedione.Phosphorylation of the extracellular-regulated kinase / (ERK/; pERK/) occurs as a downstream event to PKA activation in theca cells concomitant with CREB phosphorylation, inhibition of progesterone synthesis, and stimulation of androgens synthesis by differently modulating the transcription of genes encoding steroidogenic enzymes ().Most importantly, ERK signaling is linked to proliferation and viability in all gonadal steroidogenic cells (, ) and antiapoptotic processes (), revealing the central role of the molecule in regulating GPCR signals, including LH and hCG function, and reproduction.However, the activation of pERK/ is linked to several other intracellular processes after gonadotropin stimulation.It is required for the steroidogenic response to LH in certain cell types, such as Leydig cells (), for receptor mRNA downregulation (), and to modulate activation of GPCR kinases involved in receptor phosphorylation and subsequent internalization by b-arrestins ().The recruitment of b-arrestins, by itself, is responsible for a second pERK/ activation () as a likely opposing effect to cAMP proapoptotic events mediated by GPCRs ().Gonadotropin-induced mobilization of intracellular Ca + was first investigated in hCG-treated, transfected cells expressing the murine Lhr and was associated with PLC activation ().The signaling cascade is triggered by the Gaq protein activating PLC, with subsequent cleavage of phosphatidylinositol ,-bisphosphate to diacyl glycerol and inositol trisphosphate (IP  ).IP  binds calcium channels located in the endoplasmic reticulum, resulting in Ca + release in a hormone concentration-dependent manner.Although both cAMP/PKA and PLC/Ca + pathways are activated simultaneously, the hCG half-maximal effective dose (EC) is -fold higher for Ca + mobilization than that needed for cAMP recruitment and is independent of receptor density, demonstrating that LHCGR carries a dual signaling potential ().Ca + binds the calcium-modulated protein calmodulin resulting in downstream activation of calcium-modulated protein calmodulin kinases, which control cholesterol transport into mitochondria and steroidogenesis (, ).Moreover, Ca + signaling was associated with proliferative effects in vitro ().Simultaneous with these events, the bg dimer of G protein might lead to phosphatidylinositol ,-bisphosphate phosphorylation to phosphatidylinositol ,-trisphosphate by phosphatidylinositol-,-bisphosphate -kinase, thus activating AKT (also known as protein kinase B).AKT might also be activated through the epidermal growth factor (EGF) receptor, exerting antiapoptotic roles and inhibition of CYPA expression (, ) and, thus, negatively modulating steroidogenesis, at least partially.In contrast, activation of AKT and ERK/ signaling are necessary for STARD expression, and it is a pathway preserved across mammalian gonads and adrenal glands of mammals to mediate gonadotropin-and ACTH-induced steroidogenesis, respectively ().Taken together, gonadotropin signaling might stem from a balance between opposing steroidogenic and proapoptotic vs proliferative and antiapoptotic intracellular events providing the endocrine regulation of reproduction.
An interesting and relatively recent development is the dependence of the gonadotropin-mediated signaling on receptor concentrations, owing to preferential coupling to b-arrestin/ERK/ and AKT pathways at a relatively low receptor density as an alternative to the canonical cAMP/PKA pathway (, , ).These data provide a compelling, albeit speculative, regulatory mechanism that might contribute to differentiating gonadotropin signals, depending on the physiological requirements.LHCGR expression is a dynamic event during the menstrual cycle () and might be associated with different LHdependent roles.During the follicular and luteal phases of the menstrual cycle, the equilibrium between LH-driven proliferative and steroidogenic signals might be differentially regulated through LHCGR coupling, modulating granulosa cell proliferation, luteinization, and androgen or progesterone synthesis.At the early antral stages, LHCGR-mediated signals mainly consist of the regulation of proliferative signals delivered to granulosa cells, and these can occur as a result of unique patterns of intracellular signaling cascades, plausibly activated by FSHR-LHCGR heterodimers (, , ).The LH-dependent androgenic potential of ovarian follicles progressively increases, together with LHCGR expression levels, in theca cells during the late antral stage, suggesting that receptor density is linked to specific signaling patterns, relying on variable LHCGR concentration-dependent Gas protein coupling accompanied by increasing PLC activation () and cellular metabolic changes.These data highlight the specificity of LH as an irreplaceable ligand of LHCGR during physiological follicular phases.
Agonist-induced desensitization is a well-known feature of LHCGR and is characterized by the organization of large receptor aggregates at the cell membrane () preceding their internalization, which, in turn, is the main determinant of downregulation () regulated by GPCR kinases, b-arrestins, and other modulators ().Experimental evidence supports the formation of ligand-dependent LHCGR aggregate structures, suggesting formation of large LHCGR complexes induced by hCG rather than LH binding ().LHCGR aggregation might be a determinant for localization of a number of receptors within endosomes to determine trafficking and recycling of these molecules.Persistent cAMP signaling can be induced by the internalized receptor forming complexes with both G proteins and b-arrestins () and suggests it is likely modulated via the adaptor protein interacting with pleckstrin homology domain and leucine zipper  ().The molecular mechanism underlying persistent cAMP signaling is of great physiological relevance, because it could play a crucial role in sustaining LH functioning at midcycle in the female (), thus bypassing the potential arrest of steroidogenic signals due to LHCGR downregulation.
Whether hCG action is mediated through similar receptor features during pregnancy is unknown.A study evaluating whether the LHCGR kinetics of internalization is linked to LH-and hCG-specific treatment using fluorescent microscopy failed to find any differences in human primary granulosa lutein cells ().However, the existence of hormonespecific LHCGR trafficking cannot be excluded, because hCG displays greater potency in b-arrestin  recruitment compared with LH, at least in the transfected HEK cell line ().
The analysis of LHCGR-mediated signaling cascades provides a complex picture of intracellular events occurring with LH and hCG binding to this receptor, suggesting that hormone-specific signals might occur at different levels and result in a refined, cell-specific modulation of the biological effect.
The steroidogenic pathway Modulation of the steroidogenic response is of crucial relevance for the preservation of different physiological functions, such as gametogenesis and pregnancy, when estrogen, androgen, or progesterone variability occur as major stage-and sex-specific products.Therefore, LH and hCG might be linked to different controls of the steroidogenic pathway, regulating different and specific functions.Differences in intracellular signaling might be induced by LH or hCG binding to LHCGR, likely depending on hormonespecific conformational changes at the receptor level ().Because these spatial changes affect the intracellular portions of LHCGR, resulting in G-protein activation, it is plausible that LH and hCG treatment is linked to activation of hormone-specific patterns of signaling pathways.Mechanistic in vitro experiments, performed using bioluminescence resonance energy transfer technology and transfected cell models, revealed lower LH-than hCG-dependent levels of Gaq protein activation and intracellular Ca + increase.In contrast, no differences in Gas protein subunit recruitment and formation of dimeric or oligomeric complexes were observed ().These data indicate that the hormone-induced conformation of the receptor affects activation of LHCGR intracellular interactors and downstream signaling pathways.
Results indicative of different steroidogenic potentials related to LH and hCG were provided by the evaluation of cAMP production in human primary granulosa luteal cells (), naturally expressing LHCGR.Dose-response experiments showed that hCG is approximately five times more potent than LH in inducing cAMP production, a result exacerbated by the different EC values of the two hormones (~ pM for hCG and  pM for LH).This result was later confirmed in transfected COS- and HEK cells (, ) in the mouse tumor Leydig MLTC cell line () and in goat granulosa cells () and mouse Leydig primary cells (), in which hCG exhibited greater potency than LH, despite different EC values from those observed in human primary granulosa cells, likely indicative of cell-specific LHCGR expression and coupling to intracellular interactors.Equipotent, nonsaturating concentrations (EC) of both gonadotropins induced similar plateau cAMP levels, reached in ~ hour but, following, however, different kinetics ().In particular, the LH-induced cAMP response is rapid and reaches a plateau after  minutes.In contrast, hCG treatment requires more time, suggesting the existence of different regulatory mechanisms underlying the steroidogenesis mediated by LHCGR bound to each ligand.However, the translation of in vitro data into physiology is not immediate owing to the several perturbing factors present in vivo, which can lead to biased evaluations and affect data interpretation.For instance, the addition of FSH to LH and hCG in vitro resulted in a fivefold increase in potency of hCG in inducing cAMP production but did not have any effect on the LH-specific response in human granulosa cells ().This finding highlights the relevance of the coexistence of LH and FSH during the follicular phase of the menstrual cycle, which should not alter the FSH-mediated steroidogenic signal.However, the addition of hCG to FSH administered during COS cycles might not lead to similar effects at the molecular level.
cAMP recruitment reflects downstream pCREB activation and STARD gene expression, which is more sustained with hCG treatment, in human primary granulosa cells () and potentiated by FSH cotreatment ().The STARD gene encodes the steroidogenic acute regulatory protein enzyme modulating cholesterol transport into mitochondria, a ratelimiting step for steroid synthesis.Because these results were corroborated by the greater activity of the CREreporter gene after hCG, but not LH, stimulation, in both MLTC and transfected HEK cell lines (), the higher levels of cAMP/PKA pathway activation obtained with hCG vs LH treatment in different cell models were likely driven by intrinsic characteristics of the ligand-receptor complexes, rather than cellspecific intracellular enzymatic environments.These data, therefore, strengthen the hypothesis that hCG has greater steroidogenic potential than LH, reflecting the role exerted during pregnancy by CGs in supporting massive progesterone production.In contrast, the dual-proliferative and androgen-stimulating-roles exerted by LH in granulosa and theca cells, respectively, might not require induction of the steroidogenic pathway at the levels necessary in pregnancy.It is reasonable that androstenedione synthesis by theca cells is mainly supportive for conversion to estradiol, and its levels are similar for the whole antral phase ().Therefore, high androgenic potential should not be required to support follicle growth.Long-term ( to  hours) treatment of granulosa cells with LH resulted in greater CYPA gene expression "LH and hCG retain different steroidogenic potentials modulated in a cell-specific manner."compared with hCG treatment (), although no differences were found in the short-term ( to  hours) ().The positive action of LH on CYPA gene expression, which encodes the aromatase enzyme, is consistent with the requirement for suitable estrogen production to support ovarian follicle growth.In contrast, LH steroidogenic activity should be focused on oocyte growth by converting progesterone to androgens and eventually to estrogens and on transient maintenance of the corpus luteum, actions requiring only limited progesterone production as a basal substrate for androgen synthesis.In contrast, the role of hCG in sustaining pregnancy is exerted through massive progesterone production, which could be exacerbated in granulosa cells by high levels of cAMP/ PKA pathway activation, at least in vitro ().
Because the collection of sufficient primary theca cells and the development of stable theca cell lines () suitable for in vitro experiments are challenging, in vitro evaluation of androgen synthesis is often performed using adrenal or Leydig cell lines.In the mouse Leydig tumor-derived MLTC cell line, progesterone dose-response curves produced by LH and hCG treatment resulted in lower EC and higher plateau level achieved with hCG compared with LH treatment.However, the testosterone dose-response curves are similar and differ only from the LH and hCG EC values, reflecting the hormone potency inferred by cAMP data ().In this cell model, progesterone is a precursor of testosterone, and its synthesis is strictly connected to steroidogenic acute regulatory protein activity, the gene expression of which is activated more by hCG than by LH, thereby explaining the greater hCG-induced plateau level of progesterone.Although these data were obtained in Leydig cells, they corroborated hCG function in pregnancy, suggesting that the steroidogenic role of hCG is focused on the control of efficient, massive progesterone production, which could be useless, or even counterproductive, during the follicular phase.Instead, in the Leydig MLTC cell line, LH-and hCGmediated testosterone production was similar and reflected the cAMP data, suggesting that synthesis of the final steroid (testosterone) is regulated by quantitative stimuli and substrate availability, with no need for qualitative control of earlier events.
Scientific studies have provided several data comparing the effects of pituitary LHs and chorionic gonadotropins of humans and other mammals (, ), based on the promiscuity of glycoprotein hormones and their receptor systems across the phyla.The differences in cAMP production induced by human LH and hCG and mediated by rodent Lhr (, ) match those found in human granulosa cells (, ).However, downstream LH-and hCG-induced pCREB, Stard gene expression, and testosterone synthesis are all highly similar in mouse primary Leydig cells in vitro ().This effect might be the result of a balance between stimulatory and inhibitory signals obtained by treatment with human gonadotropins through the cAMP/PKA and ERK/ pathways.Most importantly, these data indicate that only a quantitatively, but not qualitatively, different intracellular response occurs in rodent primary Leydig cells.This likely results from their enzymatic equipment, capable of triggering testosterone synthesis such as an "on/off switch," more than in discriminating between steroidogenic and proliferative signals, such as occurs in granulosa cells.This concept might find support in observations recently described in a case report.In a hypogonadal, hypophysectomized man sequentially treated by LH and hCG (), both gonadotropins showed similar efficacy in inducing testosterone production.
The inconsistency between human granulosa and rodent Leydig cells might have resulted from differences in amino acid sequences between LHCGR and mouse Lhr.They share only % identity at the hinge region (), which is responsible for discriminating between LH-and hCG-mediated signaling (), suggesting that the evolutionary divergence between the two species impedes the qualitative discrimination of intracellular signals mediated by human hormones despite effective receptor binding.These data provide relevant insights for the so-called Van Hell in vivo bioassay (), currently used for inferring gonadotropin dosages for clinical purpose in humans by only evaluating testosterone-dependent endpoints (, ).In vivo bioassays aim to assess the biological activity, measured in terms of organ weight gain, of pharmacological preparations injected into living rodents.However, these assays might not be appropriate for detecting the full spectrum of gonadotropic bioactivity mediated by LHCGR in human cells.
Taken together, LH and hCG retain different steroidogenic potentials modulated in a cell-specific manner.Although a qualitative discriminatory capability between LH-and hCG-mediated signals is displayed by granulosa cells, steroidogenesis, mainly androgen production, in Leydig and theca cells is regulated by quantitative signals.These data highlight the specific roles for each gonadotropin in their target cells, providing experimental evidence that LH and hCG are not equivalent.

Proliferative and proapoptotic signals
Proliferative signals control antral follicle growth in the presence of both FSH and LH (), and hCG physiologically acts on LHCGR-expressing cells dedicated to progesterone synthesis.Pregnancy is also characterized by angiogenic events, consistent with cell proliferation, and possibly mediated by hCG-H, growth factors, and steroids.It is, however, plausible that LH and "classical" hCG differentially affect cell proliferation, given the specific molecular properties required to optimize follicle growth and massive progesterone production.Discrimination of qualitatively different LH-and hCG-mediated signaling by LHCGR was demonstrated in human primary granulosa lutein cells by assessing activation of phosphoproteins.Both gonadotropins showed maximum activation of ERK/ and AKT pathways at a concentration of  pM, resulting, however, in a hormone-specific phosphorylation pattern of these kinases ().LH treatment resulted in greater and more sustained pERK/ and pAKT activation compared with hCG (, ), reflecting the proliferative and antiapoptotic roles (, ) exerted by LH during the antral phase of follicular development.Moreover, the range of effective LH concentrations is widened by the presence of FSH combined with LH, but not with hCG, confirming that gonadotropin-specific activities are potentiated by FSH cotreatment ().LH and hCG displayed different ratios between the cAMP EC and the maximally activating concentrations required for pERK/ and pAKT activation (~: for LH and ~: for hCG) in granulosa cells.These data suggest that LH has greater proliferative and antiapoptotic potential than hCG in granulosa cells in vitro.This is an example of biased signaling, consisting of activation of different intracellular endpoints evoked by varying concentrations and ligands.
The expression of proliferative and antiapoptotic genes in granulosa cells reflects hormone-specific signal transduction.The expression of the growthpromoting AREG gene (), which encodes EGFsimilar amphiregulin, and of the CCND gene (), encoding cell cycle regulator, G/S-specific cyclin-D, is upregulated more by LH than by hCG.In contrast, the high steroidogenic potential of hCG, which relies on efficient cAMP production, is linked to expression of proapoptotic genes in primary granulosa cells in vitro, especially in the presence of FSH.Although both LH and hCG elicited increased TP proapoptotic gene expression, the procaspase -encoding gene CASP was positively modulated by hCG treatment, although LH treatment failed to obtain similar results, likely due to upregulation of the antiapoptotic X-linked inhibitor of apoptosis proteinencoding gene XIAP ().This finding should be confirmed in other cell types.However, gene expression data supported a dose-dependent cell viability decrease in human granulosa cells maintained for  to  hours under hCG stimulation in vitro.However, LH resulted in the opposite effect by counteracting cell death (, ).Similar conclusions were drawn from a study comparing the long-term effects of LH and hCG in goat granulosa cells in vitro.Although hCG induced relatively high cAMP intracellular levels and reduced cell viability, LH treatment resulted in marked phosphorylation of ERK/ and an increased rate of cell proliferation ().
Whether hCG is capable of mediating proapoptotic effects in vivo, at least during the follicular phase of the menstrual cycle, should be investigated in-depth, because the molecule was associated with both proliferation and inhibition of cancer cell growth (, ) and given the proliferative role exerted during placentation.It is plausible that hCG-dependent life signals are cell specific and sensitive to a particular hormone isoform or glycosylation variant ().However, the proliferative and antiapoptotic signals should be transmitted through the ERK/ and AKT pathways (, ), which are preferentially activated by LH, rather than hCG, at least in human primary granulosa cells in vitro.hCG displayed greater efficiency in recruiting b-arrestin  compared with LH, which has a -fold greater EC (hCG EC ~ nM, LH EC  nM) and even acts as a partial agonist, not reaching hCG-dependent plateau levels ().Although these data were obtained using the MLTC cell line, they are likely to also be valid in human granulosa cells, because the recruitment of b-arrestins is dependent on GPCR activation by agonists ().Most importantly, these data reflect the lower efficacy and efficiency of LH compared with hCG in inducing progesterone production, indicating that b-arrestins are involved in steroidogenesis, as demonstrated by the decreased progesterone levels on depletion of b-arrestins by small interfering RNA probes ().b-Arrestins are involved in ERK/ phosphorylation and GPCR internalization (), proliferative signals in granulosa cells ().It is conceivable that relatively high amounts of LH would be required to downregulate the LHCGR-mediated steroidogenic and proapoptotic signaling (), owing to the less efficient cAMP production induced by LH compared with hCG.These data support the idea that LH has greater proliferative potential than hCG, at least in granulosa cells, reflecting its physiological function as a follicle growth regulator (Table ).

Cross-talk between gonadotropin-and steroid hormone-mediated signaling
The effect of LH and hCG on signal transduction might be precisely revealed by in vitro experiments, in which the perturbations and interactions between signaling pathways activated by different hormones and paracrine factors are missing.Proapoptotic effects linked to the addition of hCG were poorly reproduced in ART cycles in vivo and showed weak effects or even antithetical results (, ).This concept is also valid for cancer cell growth (, ), which can be both positively or negatively susceptible to the presence of the hormone, depending on the cell type.Therefore, intracellular effects clearly dissected in vitro could be masked in vivo.
In the gonads, LH and hCG induce steroid hormone production, activating the same pattern of intracellular signaling cascades.Steroid compounds, such as glucocorticoids, have been associated with the proliferation of granulosa cells and protection against "Intracellular effects clearly dissected in vitro could be masked in vivo."cAMP-and p-induced apoptosis ().These molecules act through activation of pAKT and pERK/ counteracting intracellular death signals.
During the follicular phase of the menstrual cycle, gonadotropin functions converge in the production of progesterone, which is further converted to androgen in theca cells.Androgen is transferred to granulosa cells, where it is transformed to estrogen, leading to potent proliferative effects and inducing follicle growth in vivo.Estradiol activates the AKT pathway in vitro (), and estrogen receptor-b knockout mice are characterized by impaired follicular development ().It is reasonable that estradiol could counteract the proapoptotic signals mediated by high cAMP intracellular levels, obtained during treatment by FSH and hCG administration in COS.This concept was confirmed in vitro by adding estradiol to hCG or LH treatment of human granulosa cell cultures ().The effect of the addition of steroid predominates compared with that of either LH or hCG alone, triggering high levels of pAKT activation and preventing hCGdependent procaspase  cleavage and decreased cell viability.This molecular mechanism could explain why the addition of hCG to FSH in COS for ART has been associated with multiple follicular development, reasonably sustained by high estrogen levels and avoiding natural follicular atresia.
Progesterone is the main steroid produced during the luteal phase and by human primary granulosa lutein cells in vitro, and it is responsible for the protection from apoptosis linked to hCG treatment ().In contrast, it is well known that both LH and hCG, as well as progesterone, promote ERK/ and AKT phosphorylation.They also induce expression of AREG and the epiregulin-encoding gene EREG, two EGF-like ligands with positive effects on granulosa lutein cell viability in vivo and preservation of the corpus luteum in primates () and rodents ().The same intracellular pathways lead to the growth of progesterone receptor-expressing cancer cells (), strengthening the evidence for proliferative and antiapoptotic effect mediation by steroid hormones through activation of signaling cascades common to gonadotropins.These data support the tumorigenic potential described in hCGb-overexpressing transgenic mice, in which human gonadotropin and high levels of progesterone can assist in the development of multiple cancers (, ).However, because extragonadal tumors were totally abolished by ovariectomy in these mice, it is plausible that the tumorigenic potential of hCG is exerted through aberrant ovarian functions, rather than by a direct gonadotropin effect ().The apoptotic effect seems to be directly dampened by hCG treatment in Leydig cells (), in which the signals induced by both LH and hCG converge on a similar balance between cAMP production and ERK/ phosphorylation and downstream steroidogenesis (), suggesting the different nature of LHCGR-mediated signaling within the testis and ovary (Fig. ).

Polymorphisms and Mutations
Several single nucleotide polymorphisms (SNPs) and mutations occurring within gonadotropins and their receptor genes have been described (, ).They can modulate or impair the hormonal response or receptor function, affecting reproductive function or leading to disease.Generally, spontaneous mutations occur as random events and are silent, not resulting in amino acid changes at the protein level.Some, however, cause changes in the amino acid sequence and are linked to clinical phenotypes.In contrast, SNPs result in mild phenotypes without strong repercussions on the reproductive success of individuals.They have a frequency of $% in a given population and contribute to the endocrine ethnic background.Most SNPs and mutations affecting LH and hCG signaling are carried by LHCGR ().
Among the several SNPs and mutations occurring within the LHB/CGB gene cluster, only a few have been associated with specific phenotypes.A better understanding of the consequences of LHb and CGb mutations was provided by the study of transgenic mice models.The LHB gene knockout male mice present with hypogonadism, with low testosterone levels and hypoplastic Leydig cells, but have normal serum FSH levels; the female mice are infertile and feature anovulation and degenerated antral follicles ().The mouse phenotype partially matches the clinical observations in humans, in whom LHB gene mutations lead to a severe, eunuchoid phenotype in males ().Therefore, mouse models might not provide a full comprehension of human LH physiology.Administration of exogenous FSH is enough to sustain follicular development and ovulation in hypophysectomized female mice, and this hormone fails to do the same in Lhr knockout mice ().These findings support the concept that the presence of gonadotropin receptors, rather than LH, is required to sustain gametogenesis in mice.Transgenic mice overexpressing both the CGB and the CGA subunits were expected to replicate the human phenotype linked to activating mutations of LHCGR (i.e., asymptomatic women and men developing precocious puberty and testicular tumors).However, these mice featured the opposite phenotype, with a normal phenotype in males, and precocious puberty, obesity, and luteinized ovaries with luteomas and hemorrhagic cysts present in females ().However, although transgenic mice might not be fully representative of human physiology, their study has provided new insights to comprehend LH/hCG-and LHCGR-specific functions emerging from clinical data involving SNPs and mutations (described in the next sections: "LHb and hCGb polymorphisms"; "LHb and hCGb mutations"; and "Receptor mutations and polymorphic variants").

LHb and hCGb polymorphisms
Few LHB and CGB gene polymorphisms were studied in conjunction with their clinical phenotype.Overall, they do not severely affect fertility, and most lack molecular characterization in vitro, with no clear insights provided of their suggested effects in vivo.One exception is that provided by the most common LHB gene variant (V-LH), which was discovered in the Finnish population and displays a double polypeptide change (i.e., tryptophan to arginine at position  and isoleucine to threonine at position  of the amino acid chain) (, ) and introduces a glycosylation site (), hiding the molecule from a specific anti-LH antibody ().V-LH exhibited a reduced serum halflife and bioactivity in vivo compared with wild-type LH and decreased receptor binding activity and potency for progesterone production in vitro () and induced preferential IP  -related signaling, rather than cAMP/PKA ().Perturbation of the signaling cascade by V-LH might affect granulosa cell survival and follicle development.In cumulus cells of heterozygous women undergoing ART, high levels of apoptotic markers were found, such as the DNA fragmentation index and cleaved caspase-, and they negatively influenced the success rate of intracytoplasmic sperm injection procedures ().However, increased expression of this variant, identified by SNPs within the promoter region in linkage disequilibrium, resulting in follicular phase, granulosa cells express both FSHR and LHCGR capable of forming homodimers/ heterodimers, with theca cells expressing only LHCGR.In granulosa cells, hCG displays greater steroidogenic potential than LH, exerted via relatively high levels of cAMP/PKA pathway activation, and this feature is potentiated in the presence of FSH.hCG action is exacerbated by massive production of progesterone, which is converted to androstenedione in the theca cell, and testosterone is a minor product in the gonads of the female.Androgens serve as a substrate for the aromatase enzyme, which converts them to estrogens with high proliferative and antiapoptotic potential.Intracellular cAMP increase is linked to proapoptotic stimuli, exacerbated in vitro by the absence of theca cell-derived substrate for estrogen synthesis.LH displays lower potency than hCG in terms of cAMP/PKA pathway activation, resulting in relatively low steroidogenic and proapoptotic potential.LH signals are preferentially exerted via phosphorylation of ERK1/2 and AKT after the recruitment of G protein and b-arrestins and resulting in proliferative/antiapoptotic events.LH-specific signals are potentiated in the presence of FSH, which reasonably provides the main steroidogenic stimulus in granulosa cells.Theca cell androgenic potential increases, together with the progression of antral follicle growth and the amount of LHCGR expression, providing sufficient androstenedione to be converted to estradiol.To date, no in vitro studies have compared the action of LH and hCG in theca cells.Because Leydig cells are androgenic and exhibit LHCGR expression, they can be considered the male counterpart to theca cells.hCG is more potent than LH in inducing both cAMP/PKA and pERK1/2 pathway activation but results in a qualitatively similar balance of stimulatory and inhibitory steroidogenic signals and downstream testosterone synthesis.
~% greater activity compared with that of the "normal" LHB promoter, compensates for the weaker hormone bioactivity ().The frequency of V-LH was lower in obese women affected by polycystic ovary syndrome (PCOS) than in healthy women and nonobese women with PCOS, suggesting that V-LH might provide protection from developing symptomatic PCOS in obese women ().Nonetheless, this LH variant was associated with infertility in homozygous Japanese women (), although Baltic V-LH carrier men affected by idiopathic infertility had higher serum LH levels than healthy men ().V-LH was found in a -year-old man affected by the fertile eunuch syndrome ().This patient displayed hypogonadism and normal responses to treatment with exogenous GnRH and hCG.Overall, however, the ethnicityrelated clinical features displayed by V-LH and the mild phenotypes suggest that it simply represents an example of phenotypic variations due to genetic polymorphisms as the basis of human diversity.An SNP (rs) in exon  of the LHB gene was found more frequently in South Indian women with PCOS ().This SNP is characterized by the synonymous amino acid change "T" to "C" at position  of the gene sequence.Its contribution to PCOS pathogenesis is unexplained.It is conceivable that the polymorphism might affect the expression or functions of other molecules, because it is within the palindromic RUVBL gene sequence, encoding for a protein interacting with the activating transcription factor  ().Several other SNPs within or close to the LHB gene sequence were associated with central precocious puberty () or infertility () in women.However, their contribution in defining the endocrine phenotype should be evaluated with other polymorphisms related to hormones and their receptors.Moreover, these results should be independently confirmed in other populations and supported by functional in vitro data, which are still lacking.
Among the CGB gene SNPs and mutations previously described in association with miscarriage () and discussed in the section "hCGb isoforms and glycosylation variants," a polymorphism within the CGB gene displayed inefficient assembly when cotransfected with the CGA gene in the CHO cell line.This naturally occurring variant is associated with infertility in women and is characterized by a valine to methionine exchange at codon  ().Seventy-one hCGb variants deriving from CGB and CGB genes might be predictive of recurrent miscarriage in European populations (), suggesting the relevance of these transcripts in sustaining pregnancy.Moreover, because the heterozygous haplotypes calculated for these SNPs are relatively frequent, they might be subjected to balancing selection in Europeans.Overall, the associations between CGB gene SNPs and miscarriage are relatively rare, and the molecular mechanisms supporting the clinical data are unclarified.
In general, such as in the case of SNPs and common variants in other genes, SNPs in LHB and CGB genes are unlikely to be major determinants of disease.Rather, they contribute to human phenotypic variation and, together with other SNPs, might be relevant in discrete genomic clusters associated with particular reproductive problems, such as PCOS ().

LHb and hCGb mutations
Possibly because of the physiological role played in a specific time-window and in one sex only, inactivating hCG mutations are rare and, presumably, incompatible with successful pregnancy.One of the first mutations affecting hCG function was somatic and was found in the a subunit secreted by undifferentiated carcinoma cells of the femoral region ().This mutation consists of a substitution of glutamic acid by an alanine at position , which changes the hydrophobic profile of the molecule, resulting in misfolding and impairment of dimerization with the b subunit.Similar effects at the molecular level have been associated with a genomic CGB gene mutation, leading to a valine to leucine swap at position  of the b subunit ().The mutated hCGb is only capable of % dimeric assembly yet shows increased potency in inducing cAMP production.Nevertheless, enhanced steroidogenic signaling was not sufficient to replace the loss of function due to impaired dimerization, because this mutation was found in a Northern European patient affected by recurrent miscarriages.CGB gene mutations have also been described and found in individuals of the same population (), albeit without a link to pathogenic phenotypes.A proline to arginine change in position  resulted in a twofold reduction in b-subunit secretion without affecting its biological activity, and no effects were associated with an arginine to tryptophan substitution at position .Altogether, these data indicate that genomic mutations within the genes encoding for the two major hCG transcripts result in mild consequences that can be tolerated.
Although mutations in the CGB genes predominantly result in miscarriage, mainly due to misfolding of the b subunit and impaired dimer formation (), LHB gene mutations result in phenotypes featuring hypogonadism, decreased or impaired spermatogenesis, delayed puberty, and low testosterone levels in males and amenorrhea in females.Such symptoms might be associated with infertility in both sexes and might be due to disruption of LHB splice sites () or in the signal peptides ().Mutations disrupting the cystine knot motif, such as a glycine to aspartic acid mutation at position  might result in defective heterodimer formation, undetectable serum LH levels, and hypogonadism ().A similar phenotype was described in a patient carrying a lysine deletion at position  of LHb, which impaired release of hormone dimers ().Because mutations within the LHB gene do not impair receptor function, long-term administration of exogenous hCG to these patients induces virilization, testicular growth, testosterone synthesis, and spermatogenesis, in conjunction with fertility ().These data strengthen the hypothesis that LH and hCG could be equivalent in inducing proper Leydig cell function and fertility, which mainly relies on testosterone synthesis in males and might be switched on by an on/off molecular mechanism, rather than qualitatively different hormonal signals ().However, it cannot be excluded that signaling cascades are maximally activated by low amounts of occupied receptors, confirming previous findings in rodent Leydig cells in vitro ().Certain mutants of LHb might display residual in vitro activity, sustaining low testosterone synthesis but enough to support normal spermatogenesis and fertility ().Studies of siblings carrying the same LHB mutations that totally disrupt LH signaling revealed different, sexspecific clinical effects.Although normal pubertal maturation might be conserved in women, this is not the case in men (), suggesting that, in women, the regulation of estrogen production needs to be sustained by FSH acting on granulosa cells, supported by basal androgen production originating from theca cells.Moreover, in the ovary, LH-mediated signals might plausibly be driven by LHCGR-FSHR heterodimers activated by FSH at the onset of puberty and during early antral follicular stages (), although Leydig cells are the only LH target and source of the proper testosterone levels required to support male secondary sex characteristics.Nevertheless, inactivating mutations of LHb subunit have been associated with ovulatory disorders (), suggesting the fundamental relevance of proper LH signaling for the ovarian cycle.

Receptor mutations and polymorphic variants
Although several SNPs were found within the LHCGR gene, their contribution in determining clinical phenotypes is mostly weak.An exon  SNP, p.NS (rs), was found in association with spermatogenic damage and is highly prevalent among infertile male patients ().However, although the number of LHCGR marker SNPs affecting male reproductive function is very low, it is remarkable that the very gene is a genetic hot spot for PCOS in the Han Chinese population ().
A common polymorphism of LHCGR consists of the addition of two amino acid residues, a leucine and a glutamine, at codons  and  within exon , originating from a CTCCAG insertion at positions - of the gene ().This receptor variant, known as "insLQ," is common among white populations but absent in other populations, such as the Japanese.Although the double amino acid residue insertion does not severely affect LH-and hCG-mediated cAMP production, the polymorphism has been linked to a more active signal peptide and to adverse outcomes in patients with breast cancer ().
The LHCGR gene mRNA variants comprise highly expressed, primate-specific transcripts, including a cryptic exon located between the sixth and seventh exon with unknown function ().This exon is named "A" and is responsible for three different mRNA receptor variants differing in the length based on the location of stop codons.The resulting mRNA variants, in addition to the "classical" LHCGR, include two truncated forms consisting of exons  to  and one A of different length and a full-length transcript with exon A between exons  and .Mutations within this region are causative of aberrant gene transcription, leading to Leydig cell hypoplasia type II ().In addition, SNPs in exon A were associated with testosterone levels in male infertile patients ().In particular, the levels of this hormone were more elevated in G homozygous men than in those carrying the T allele of the exon A SNP rs.Mutations of this cryptic exon were discovered for first in ,XY patients affected by sex development disorders and characterized by a female phenotype, a blind-ending vagina, and primary amenorrhea ().Based on current knowledge of human testicular development, the presence of testicular structures and a phenotype largely resembling testicular feminization (which is due to the lack of androgen action), suggest that the patient was not at all responsive to endogenous LH and maternal hCG.Therefore, it is plausible that isoforms deriving from exon A could be linked to the discrimination between hCG-and LH-mediated signals.Because our knowledge of the control of human testicular development by fetal pituitary LH or maternal hCG is limited and provided by individuals affected by genomic mutations and data from nonhuman mammals, the putative role of exon A in discriminating LH and hCG remains unclear.
Interesting data on LH-and hCG-specific functions were provided by the deletion of the LHCGR exon -encoded sequence, described in the section "Evolutionary convergence: trophoblast LH and pituitary CGs."As discussed, the mutated receptor is capable of transmitting hCG, but not appropriate LH, signals, despite binding both hormones (), resulting in male hypogonadism with a normal male phenotype ().The clinical evidence derived from naturally occurring mutations or deletions of LHCGR exons A and  has demonstrated that these sequences of the receptor are essential for LH and hCG action and could be instrumental in discriminating between the two hormones, although the mechanism remains largely unknown.

Pathophysiology of LH and hCG
Specific profiles and levels of LH and hCG could be associated with pathological conditions, such as "Extragonadal action of LH and hCG might produce clinical effects."hypogonadism, cancer, and endocrine disorders.These effects could result from excessive or low hormone activity, altering normal physiology and leading to a wide range of clinical phenotypes.Because gonadotropins modulate cell growth, it is reasonable to assume that their action is linked to the proliferation of cancer cells and tumorigenesis.Moreover, extragonadal action of LH and hCG might produce clinical effects.For instance, the pregnancies of fetuses affected by trisomy  are typically characterized by high levels of hCG in the maternal serum, despite the low hCG synthesis capability by the placenta ().This apparent paradox might be caused by high activity of sialyltransferase- and fucosyltransferase- enzymes in trisomy  trophoblast cells, resulting in highly glycosylated and acidic hCG molecules, displaying reduced activity in vitro.hCG synthesis peaks at ~ weeks in trisomy  pregnancies and declines to lower levels than in chromosomally normal pregnancies.Regardless, because no association between sialylated hCG isoforms and trisomy  pregnancy was found (, ), although fucosylation was not investigated, any conclusions should be interpreted carefully.However, highly glycosylated molecules are more persistent owing to their increased half-life ().Moreover, high hCG levels during pregnancy could induce aberrant expression of LHCGR in the adrenal glands of the mother, resulting in the increased risk of adrenal hyperplasia and transient Cushing syndrome due to cortisol release in response to the hCG-induced signals ().Several other clinical effects related to aberrant LH and hCG signaling (i.e., hypogonadism, precocious puberty, PCOS, miscarriage, and cancer, are described in the following subsections).

Hypogonadism and precocious puberty
Hypogonadism is a pathology characterized by decreased gonadal activity and hormone production and can be caused by impairment of LH-mediated signaling ().In males, the disease can arise from the testis (primary) or from dysfunction of the hypothalamic-pituitary unit (secondary or central hypogonadism) (), although the cause for several patients with hypogonadism will remain idiopathic, suggesting a possible polygenic nature ().The phenotype associated with an impaired HPG axis might vary depending on the severity of the disease.Most severe cases can be linked to rare autosomal recessive conditions, such as inactivating LHCGR mutations, typically interfering with the development of male external genitalia and testicular descent and resulting in phenotypically female, ,XY patients with Leydig cell hypoplasia type .The disease of these patients will be unresponsive to both endogenous LH and exogenous hCG administration and is characterized by primary hypogonadism and sexual differentiation disorder, featuring the absence of Leydig cells, the lack of masculinization and pubertal maturation with a female-like phenotype and external genitalia.Milder phenotypes could be linked to LHB mutations affecting hormone function, resulting in infertile individuals with male external genitalia and maldescended testes, a micropenis, and/or hypospadias.Given the presence of a functional LHCGR, hCG therapy showed efficacy in inducing testosterone production and might restore fertility.The clinical picture is strictly connected to testosterone levels, insufficient to support male sexual development.The total blockade of Leydig cell function is largely attributed to mutations impairing receptor transport to the cell membrane () and activation of the G-protein-dependent signaling cascades ().However, mutations affecting LH binding have also been reported ().An inactivating homozygous mutation was found in a ,XY hypogonadic patient with delayed puberty, a normal prepubertal male phenotype, and descended testes, displaying a glutamine to arginine substitution at codon  of LHb ().The resulting b subunit was capable of forming a heterodimer with the a subunit but failed to bind the receptor in vitro.This patient was treated with longterm hCG administration, resulting in testicular enlargement, virilization, and spermatogenesis.Similar phenotypes and histories were found in consanguineous, hypogonadic patients bearing the deletion of lysine at position  of the LHb, linked to intracellular retention of the hormone ().These data allow for the comparison of the effects of mutations impairing LHCGR or LHb functions in the male.Although the first are linked to severe, female-like phenotypes, LHb mutations are compatible with the male phenotype and exogenous hCG-induced spermatogenesis ().Taken together, these findings suggest that maternal hCG, together with the presence of intact LHCGR, can, at least in part, compensate for the absence of fetal pituitary LH and support the development of a male phenotype, although the presence of maldescended testes and a micropenis suggest that proper production of functional LH molecules is required to fully support secondary sex characteristics.
Inactivating mutations within the LHB gene have been described in infertile females, in whom the phenotype displayed normal external genitalia and spontaneous breast and pubic hair development at puberty.Menarche was delayed or even normal; however, these patients were oligo-amenorrheic and characterized by failure in achieving ovulation and normal LH, estradiol, and progesterone levels in the ovulatory or luteal phase.Because women with LHB mutations have functional LHCGRs, they can successfully be treated with LH or hCG (), in contrast to hypogonadic women with LHCGR-inactivating mutations, which will be unresponsive to gonadotropin treatment.
Precocious puberty is defined as the onset of puberty at a relatively young age and can result from several factors, such as hypothalamic or pituitary dysfunction, McCune-Albright syndrome, or sex hormone-secreting tumors.LH signaling is one of the factors regulating the onset of puberty.However, although LHCGR-activating mutations resulting in sustained tonic cAMP production due to an aberrant ratio between the Gas (stimulating) and Gai (inhibitory) protein activation (), were linked to familial, male-limited, precocious puberty, no phenotype has been observed in females.No LHb mutations are known to cause excessive LH activity.

Polycystic ovary syndrome
PCOS is a common endocrine disorder affecting % to % of women worldwide and defined as the coexistence of at least two of three typical features: polycystic ovaries, ovulatory dysfunction, and hyperandrogenism ().Other symptoms can occur in association with the disease, such as type  diabetes, metabolic syndrome, adrenal dysfunction, obesity, and/or insulin resistance ().Because PCOS has likely maintained an overall constant prevalence over centuries, albeit linked to anovulatory subfertility, it has been addressed as an evolutionary paradox (, ).Intralocus sexual conflict was proposed to explain the persistence of genetic loci linked to increased reproductive success in males in conjunction with a risk of developing the disease in females (, ).PCOS has a polygenic nature, and genome-wide association studies found both FSHR and LHCGR genes to be two of several hot spots for the disease (, ), suggesting the relevance of gonadotropin signaling in its pathogenesis.However, endocrine-disrupting chemicals modulating sex hormone-dependent signals might be associated with the disease ().Hyperandrogenism is a major determinant of the disease and is indicative of excessive androgens produced by theca cells exposed to relatively high LH levels.Moreover, the androgen and estradiol response to FSH stimulation is greater in those with PCOS than in healthy women, suggesting that molecular mechanisms regulating paracrine signals between granulosa and theca cells are amplified in individuals carrying the disease ().As a consequence of the high estrogen levels, the feedback mechanism regulating pulsatile gonadotropin production is altered in women with PCOS, resulting in low FSH levels, a high LH/FSH ratio, and impaired follicle selection and ovulation ().In humans, cyclic gonadotropin production recovers after treatment with the estrogen receptor antagonist clomiphene citrate, which temporarily restores proper feedback mechanisms at the pituitary level ().Polycystic ovaries and adrenal disturbances are recapitulated in the phenotype of transgenic, LHoverexpressing female mice, supporting the role played by this hormone in PCOS pathogenesis ().hCGoverexpressing female mice displayed a slightly different ovarian phenotype, characterized by multiple corpora lutea and enhanced estradiol, progesterone, and testosterone levels, in addition to prolactin-linked adenomas ().Different phenotypes of female mice overexpressing LH and hCG strongly support the view of a different in vivo action of these two gonadotropins in rodents.
Appearance of the polycystic ovary is one of the characteristics that can be used for the diagnosis of PCOS and consists of the recruitment of several follicles reaching the antral stages without completing maturation.Provided that LHCGR is of central importance for the understanding of the disease, theca cell androgenic function and granulosa cell proliferation are modulated by different genes (), such as DENNDA, INSR, and RABB, whose functions are involved in modulating proliferative signals through the activation of AKT and ERK/ pathways.The activity of these kinases is increased in the ovary of women with PCOS (), compatible with exaggerated LH-and estrogen-dependent stimuli.SNPs within the LHB gene might also be linked to increased PCOS risk.This is the case for the two SNPs inducing a tryptophan-to-arginine change at position  and an isoleucine-to-threonine change at position  of LHb (), which are in linkage disequilibrium and might contribute to elevated testosterone levels in Brazilian women with PCOS.Moreover, it was suggested that the V-LH variant might have been a contributing factor in the development of the disease in a Japanese woman (), although another study failed to find a similar association in Turkish women ().This issue should be further investigated and independently confirmed in other populations.Nonetheless, it is suggestive of altered functioning of proliferative and androgenic LH activity.Most importantly, the glycosylation profile of LH molecules might change depending on the steroid hormone levels, influencing gonadotropin activity ().Although the molecular mechanism underlying the control of biologically active LH isoform production by the pituitary is unknown, age-specific profiles of LH glycosylation were reported among women with PCOS.Mainly alkaline LH species were found in adult patients with PCOS (), although basic isoforms featuring high in vitro activity were predominant among adolescent girls affected by the disease and positively correlated with androgen levels ().It is plausible that highly bioactive LH isoforms will result in elevated androgen levels, and this hypothesis is consistent with the inhibition of steroid synthesis by GnRH antagonists in women with PCOS ().
Given their chronic overexposure to "LH activity," women with PCOS will require particular attention when ovarian stimulation by exogenous gonadotropins is required in the framework of ART.Concurrent with the presence of high estradiol levels and the development of excess antral follicles, which can lead to an excessive response to gonadotropin stimulation "Women with PCOS will require particular attention when ovarian stimulation by exogenous gonadotropin is required in the framework of ART." (), women with PCOS might have an increased risk of developing ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies with ART ().Thus, ovulation induction with a GnRH agonist has been proposed as a better choice than hCG (, ), consistent with the high steroidogenic potential of the choriogonadotropin, which could be linked to OHSS.However, the matter is still under debate, and the clinical symptom spectrum related to PCOS, together with ovarian morphology, might be a determinant for the response to hCG exposure (), even in pregnancy, when the levels of endogenous-free hCGb molecules differ between healthy women and those with PCOS ().A clinical study comparing ART between white and Yoruba women from Western Nigeria, triggered by hCG, revealed higher estradiol levels and the prevalence of symptoms typically related to PCOS among African women, who have a greater risk of OHSS and twin pregnancies.This finding suggests that ethnicity could be a determinant for PCOS.The anovulatory condition linked to the disease might lead to maintenance of a follicular reserve for a longer time compared with healthy women, and this concept has been corroborated by the converging effects of genes regulating the age of menopause and LH levels ().PCOS could therefore be viewed as an evolutionary strategy to prolong the fertile window at the cost of fewer ovulatory cycles ().Although suggestive, this hypothesis should be examined further, because no evidence of evolutionary advantages was experimentally demonstrated and subfertility linked to PCOS might result in substantial effects in modern times.Today, especially in Western societies, fertility and pregnancy are sought at a relatively late reproductive female age (), and ovarian stimulation might be performed in the framework of ART using commercial hormone preparations, a setting quite different from a natural cycle in a nonmedicalized society.

Miscarriage
Most miscarriages occur within the first trimester of pregnancy and are mainly due to placental or fetal abnormalities.Embryo aneuploidies are typical causes of miscarriage () and other clinical conditions, such as diabetes and obesity, could be risk factors for poor pregnancy outcomes ().These data point to the importance of proper metabolic and endocrine function in regulating embryo implantation and development.The action of LH is central in follicle growth and uterine preparation, and hCG is required to support pregnancy, with both these aspects affecting pregnancy success.A dual function could also be assumed for LH action.It is linked to estrogenmediated proliferative events in the uterine epithelial cells during the follicular phase, resulting in increased endometrial thickness, and it becomes mainly steroidogenic during the luteal phase, when the increase in progesterone levels is associated with the secretory action in the uterus.These changes in steroidogenic potential are accompanied by the to -fold increase in LHCGR expression, from the early antral stage through to ovulation (), and are very intense in the corpus luteum ().Antral follicle growth requires proliferative signals preferentially and directly exerted by LH, as demonstrated in vitro, and estradiol, which increases via the cAMP/PKA pathway, together with the receptor number.Thus, estradiol can be used as a marker for successful pregnancy outcomes ().The maintenance of large LHCGR numbers in the corpus luteum reasonably predisposes later, massive progesterone synthesis induced by LH and, in the case of pregnancy, hCG.Thus, LH and hCG should ensure proper progesterone levels, which, in turn, will mediate the events preparatory to, and supportive of, pregnancy.Women experiencing recurrent miscarriages might achieve better pregnancy outcomes through progesterone supplementation ().These data illustrate the role of both LH and hCG in endometrial preparation and maintenance of the corpus luteum through adequate steroid production.It is reasonable that endometrial preparation by the addition of LH in ART cycles might provide adequate stimuli leading to lower pregnancy loss (), although hCG could not provide similar activity and leads to different effects in pregnancy outcomes ().As the hormone of pregnancy, hCG exerts its specific functions during the first trimester, resulting in angiogenetic effects and increased progesterone synthesis.These roles are irreplaceable, as demonstrated by reports describing the association between recurrent miscarriage and mutations of the hCGb-encoding genes, which impair signal transduction and alter the biochemical properties of the molecule ().In women with a successful pregnancy, the serum and urinary levels of the hormone will be different than those measured in women with a miscarriage ().Thus, specific hCG functions and support of embryo development also depends on hormone levels.

Cancer
Because LH and hCG might activate intracellular signaling cascades regulating cell proliferation and antiapoptotic events, it was suspected that they might retain tumorigenic potential and be involved in cancer formation via LHCGR-induced signals ().The study of mouse models revealed that tumorigenesis at gonadal and extragonadal sites is related to excessive gonadotropin levels ().Similar conclusions have been suggested by the study of CGB gene mutations, which lead to aberrant hCG production and have been linked to gestational trophoblastic neoplasia ().An interesting case report provided evidence of hCG-dependent tumorigenesis.A woman undergoing chemotherapy for metastatic renal cell carcinoma became pregnant during the treatment break period and experienced dramatic growth of the tumor ().This event was consistent with hCG production and LHCGR expression in cancer cells.In addition, clinical abortion coincided with rapid tumor regression, suggesting that the angiogenic potential of high hCG was proportional to the hormonal levels.A relationship between cancer cell growth and the production of hCGb and hCG-H molecules could be suspected (), because they are the major isoforms produced during the first weeks of pregnancy.Their tumorigenic potential might rely on the ERK/ and AKT pathways, which is surprising because these pathways are preferentially activated by LH rather than hCG.Overall, hyperglycosylated hCG molecules were suspected to induce proliferative signals required to enhance maternal immune cell modulation, embryo implantation, and trophoblast invasion at the early pregnancy stages ().In vitro findings confirmed the proliferative potential of hCGb and hCG-H ().These data are consistent with the hypothesis that hCGb and hCG-H exert intracellular signaling different from "classical," dimeric hCG () and that their action is focused on activation of cAMP/PKAmediated steroidogenic signals (, ), fundamental for massive progesterone production during pregnancy.This molecular mechanism could be the basis of a protective effect against breast cancer (), because relatively high intracellular cAMP levels could trigger the activation of apoptotic processes in certain cell types (, ), including breast cancer cells () and testicular germ cell tumors ().Moreover, high LHCGR expression levels might result in increased sensitivity to hCG-induced proapoptotic signals, suggesting that receptor levels are a prognostic value, at least for some ovarian cancers ().In contrast, hCG steroidogenic potential might be transposed in vivo by increased production of estrogen metabolites and growth factors displaying proangiogenic and proliferative activity () and, thus, indirectly inducing variable effects, depending on the enzymatic milieu of the target cells.
"Overload" of steroidogenic, LHCGR-induced signals could also be provided by excessive LH activity.The data from mouse models overexpressing human LH has proved that gonadotropin tumorigenic potential is a result of hyperstimulated ovaries, which are induced to produce high estrogen levels ().Similar results were observed in hCG-overexpressing mice (), demonstrating that, in rodents, high levels of both human gonadotropins produce comparable steroid-dependent effects in vivo, as suggested using mouse Leydig cells in vitro ().Gonadectomy of certain mouse strains has been linked to Lhr-and estrogen-independent adrenal tumors, which are phenotypically different from those developed in mice overexpressing LH, hCG, or Fshr (, ), suggesting that the adrenal gland might be a target of gonadotropin action ().These data are suggestive of molecular mechanisms putatively linking adrenal tumors and the high LH levels encountered during human menopause.A gonadotropin-responsive adrenocortical adenoma was reported in a menopausal woman, with relatively high testosterone levels in response to stimulation of cancer cells by endogenous LH ().Similar effects might arise in LHCGRoverexpressing adrenal tumors, which were found in some cases involving menopausal and pregnant women ().Large amounts of receptors would trigger aberrant activation of the Wnt pathway by binding endogenous gonadotropins, stimulating differentiation of adrenocortical cells.In addition, similar tumorigenic effects might be due to activating LHCGR mutations in males, providing high basal cAMP and inositol phosphate levels ().In such cases, both excessive testosterone synthesis and proliferative signals might be linked to Leydig cell tumors and to prostate cancer, and downregulation of the HPG axis by GnRH antagonists might be applied as clinical treatment to counteract aberrant cell growth ().
In conclusion, causality between gonadotropins and cancer is not sufficiently supported by the scientific data.Tumorigenesis might instead be linked to the action of steroids, which, in certain cases, are synthesized by tumor cells and induce proliferative events.In contrast, cancer cells can display aberrant transcription of gonadotropins and their receptor genes, as well as other hormones and receptors (), such as steroids, GnRH, growth factors, and others.In particular, the presence of a specific receptor expression per se might not be indicative of cell function, because the opposite effects could depend on quantitative expression of GPCRs ().Given the tumorspecific nature of the expression of these factors, the resulting effects are unpredictable.The complexity of tumor cell metabolism provides a unique picture that should be evaluated on a case-by-case basis, with the specific treatment choice entrusted to clinician decision.

Clinical Applications
Gonadotropins are clinically used in both sexes when endogenous production is impaired, such as in the case of HPG disruption.In hypogonadotropic (e.g., secondary or central) hypogonadism, gonadotropin administration represents the most physiological therapeutic approach.The administration of steroids (i.e., estradiol and/or progesterone in women or testosterone in men) is preferred in selected cases.However, when physiological HPG activity is to be restored and direct stimulation of the gonads is necessary, gonadotropin administration becomes mandatory.This therapy consists of daily or weekly subcutaneous or intramuscular injections of biological compounds, which are more expensive than steroids and cumbersome for the patient.Despite these "The complexity of tumor cell metabolism provides a unique picture that should be evaluated on a case-by-case basis."challenges, this is the only therapeutic approach that can mimic the complex balance of gonadotropin stimulation of the gonad.
Although the most frequent gonadotropin application is in COS for ART, this approach remains largely empirical and not sustained by strong scientific evidence.Moreover, the business related to this treatment makes the current pharmacological schemes for COS driven widely by industry, rather than by science.No scientific interest has been evident in establishing the best gonadotropin combination in clinical practice during the past few decades.However, HH, characterized by the lack of endogenous gonadotropin secretion, represents the best in vivo model to compare the different actions of gonadotropins to evaluate their kinetics and efficacy.In this section, we have assessed the current LH/hCG biological compounds and their clinical applications.
Urinary and recombinant preparations LH and hCG are both available as registered drugs (Table ).Their biological activity (i.e., calibration) is evaluated by in vivo bioassays, assessing the gonadotropin effects in living rats or mice ().The Van Hell bioassay is the standard method in pharmacopeia to assess gonadotropin bioactivity ().This method, developed in the s, is based on the daily subcutaneous injection for  days of a fixed gonadotropin dose in to -day-old immature male rats until the final measurement of seminal vesicle weight gain ().Both LH and hCG are calibrated using the Van Hell method, comparing their bioactivity to an international standard ().This method has two main limitations.First, the animal model used is unable to discriminate between LH and hCG.Second, this bioassay evaluates gonadotropin steroidogenic activity and not the full spectrum of molecular actions ().Mouse Leydig cells were recently used in vitro to detect biological differences between LH and hCG.In addition to the qualitative differences previously demonstrated in human granulosa cells (, , ), LH and hCG resulted in quantitatively different early intracellular actions of the cAMP/PKA pathways and steroidogenesis ().These results suggest that the Van Hell method might not evaluate gonadotropin bioactivity correctly, and certainly not fully, because it only evaluates testosterone-dependent endpoints (, ).Thus, calibration of LH and hCG compounds using the Van Hell bioassay does not consider the differences in molecular action described.
Historically, the first gonadotropin isolation dates back to the third decade of the th century, when two compounds with FSH and LH activities were extracted from the urine of pregnant and postmenopausal women and named prolan A and B, respectively ().Only in the s were the early gonadotropin compounds produced, first from human pituitaries and then from urine when the human menopausal gonadotropin (hMG) was purified from postmenopausal women ().These first compounds showed a FSH/LH activity ratio of :, ensured by both LH molecules and supplementation of hCG derived from the urine of pregnant women.However, a large amount of impurities was detected, consisting of LH subunits, growth factors, glycoproteins, binding proteins, and immunoglobulins ().All these residues caused high batch-to-batch variability and could influence hMG biological action, reducing the efficacy and exposing patients to possible adverse events ().The urinary purification process was subsequently improved, eliminating all residual extraneous activities but leading to progressive LH loss.To maintain the FSH/LH ratio :, LH activity was replaced by urinary hCG ().Thus, in highly purified hMG compounds, the impurity percentage decreased to %.hCG molecules represent % of the remaining protein content ().Hence, hMG compounds with the greatest purity were defined as those containing the lowest amount of LH and the greatest hCG concentration ().However, hCG molecules added to hMG are calibrated in vivo in rodents against an LH standard.Thus, the amount of hCG in the final compound is the number of molecules capable of producing a biological effect equivalent to that of the LH standard.This process depends largely on the half-life and does not consider the molecular differences between LH and hCG.Therefore, the number of hCG molecules in the final preparation could be significantly lower than the number of LH molecules needed to obtain the same biological action, with a disequilibrium, in molar terms, between receptor and ligand.This concept must not be overlooked, because the different number of LH/hCG molecules competing with the same number of receptors could be relevant in terms of LHCGR occupancy and activation ().
Overall, although FSH is easily obtained by urine purification, LH is lost during the chromatography steps, reducing the efficiency of obtaining urinederived LH preparations.In contrast, urine hCG (u-hCG) preparations were readily developed because of the abundance of hCG in the urine of pregnant women ().Thus, urine-derived biological compounds containing LH alone were not available until the advent of DNA technologies.Only urine hCG (u-hCG) compounds have been used to obtain LH activity in clinical practice.Recombinant DNA technologies led to the production of gonadotropins in CHO cell lines, with high rates of safety and consistency.Currently, six recombinant gonadotropins are available: follitropin a, follitropin b, follitropin g, lutropin a (r-LH), chorionic gonadotropin a (r-hCG), and corifollitropin a ().r-LH and r-hCG possess greater purity than the urine-derived counterparts and consist of a mixture of isoforms exhibiting a high degree of glycosylation heterogeneity, structurally and biologically comparable to that of endogenous gonadotropins, with slight differences due to posttranslational modifications ().
In addition to classical gonadotropins, recombinant DNA technology has allowed for the creation of new chimeric compounds, such as long-acting FSH (corifollitropin a) ().In this case, the FSH b subunit was coupled to the CTP of the hCG b subunit (, ).Corifollitropin shows in vitro and in vivo pharmacological activity comparable to that of follitropin g but with a longer half-life (~ hours) ().This in an interesting example of how DNA technologies could mix several gonadotropin features in the same compound.

Different clinical effects: ovarian stimulation, luteal support, and other aspects
The gonadotropin administration in COS during ART is the current in vivo model in which the different clinical effects of biological compounds containing LH and hCG can be evaluated.In this setting, although a truly standardized approach does not exist, FSH administration is always provided to obtain multifollicular growth ().In addition to this, LH or hCG administration can be added to support FSH actions, mimicking the physiological concerted action of LH and FSH.LH physiologically regulates follicular growth, stimulating theca cell production of androgens, which, in turn, serve as a substrate for estradiol production in granulosa cells ().In the late follicular phase, the estradiol increase acts through a positive feedback mechanism causing the LH surge needed for ovulation ().Finally, LH stimulates progesterone production from the corpus luteum, which is then maintained by hCG if pregnancy occurs.To mimic this complex gonadotropin-mediated process during COS, regulatory agencies have allowed for the addition of LH, hCG, or hMG to FSH, because they are still considered equivalent in clinical terms.No specific guidelines are available to select which patient might benefit from, and should be treated with, a specific combination.The selection remains largely empirical, if not arbitrary.
In contrast, hCG is generally used at the end of the COS phase to trigger final oocyte maturation.Physiologically, however, it is LH that is responsible for final follicular maturation and follicle rupture with oocyte expulsion from the follicle ().Despite this difference between COS protocols and physiology, mainly due to the historical availability of hCG but not of LH, the final ART outcome did not change when u-hCG or r-hCG was compared with LH ().Thus, no specific evidence supports the use of LH instead of hCG for triggering ovulation.In , the use of a GnRH agonist to induce an endogenous LH surge sufficient to trigger ovulation was proposed ().This alternative to hCG was not exploited until the use of GnRH antagonists for pituitary downregulation in COS, with a shorter and reversible action compared with the agonists (, ).Thus, with GnRH antagonists, the pituitary gland remains responsive and a single GnRH agonist bolus is sufficient to displace the antagonist, activate the receptor, and induce endogenous LH release, mimicking the physiological midcycle gonadotropin surge ().This surge consists of a short ascending limb of ~ hours and a long descending limb of ~ hours ().This pattern is slightly different from the physiological midcycle surge, which lasts  hours and shows three consecutive phases ().Although GnRH agonist triggering could be more physiological and could reduce the adverse event risk (, ), neither large retrospective studies () nor randomized clinical trials (RCTs) (, ) found differences between GnRH agonists and hCG for triggering ovulation.Thus, specific evidence favoring one or another ovulation trigger scheme is not available for the final ART outcome.In particular, specific hCG actions are obtained with this treatment, such as sustained progesterone production, angiogenesis promotion, blood flow and nutrition to the fetus (), umbilical cord development, and uterine growth synchronization ().Thus, hCG substitution by LH or a GnRH agonist for triggering results in the loss of all these essential hCG activities after fertilization.Thus, when a GnRH agonist trigger is used, appropriate luteal support with progesterone and estradiol should be considered ().Although this issue has not been fully clarified and needs further investigation, it has been established that luteal support with progesterone is used in all cycles.Some investigators proposed modified luteal support when a GnRH agonist trigger is chosen, using hCG either in one -IU single bolus () or in repeated to -IU boluses (), or with the addition of LH ().However, a recent meta-analysis showed that GnRH agonist trigger use, together with luteal support by LH activity, leads to similar ART outcomes compared with clinical protocols using the hCG trigger ().However, the analysis showed notably high heterogeneity in the results, limiting its clinical significance.
COS represents the best in vivo example in which gonadotropin combinations are used in pharmacological doses, although the various protocols have not been based on physiology or sound scientific evidence.LH and hCG have been used interchangeably in this "unphysiological" context, and it is surprising how well ART has worked despite the sometimes disparate and "creative" stimulation protocols.It is difficult to draw physiological conclusions about LH and hCG from the supraphysiological setting of COS.HH should represent a better model to understand the different effects in vivo of LH and hCG, if they exist.
Clinical experience with LH and hCG: the present and the future LH activity is needed in clinical practice in the management of both female and male hypogonadism when estrogen and androgen production should be stimulated through the direct action on theca and Leydig cells, respectively.Although both LH and hCG are currently commercially available, historically, only hCG has been used, because it was the only readily available LHCGR ligand.In theory, both LH and hCG could be used now despite the complete lack of scientific evidence in favor of one vs the other.This is true in many countries for female hypogonadism, but not for male hypogonadism, because only hCG administration is permitted by regulatory agencies for the latter.It has been widely demonstrated that hCG is efficient at restoring eugonadism in HH men.In the case of male hypogonadal hypogonadism, the standard therapeutic approach has been based on hCG administration since the s ().In this setting, convincing evidence about the efficacy of hCG administration is available, with different dosages and schemes, ranging from  IU every other day () to $ IU two times weekly ().However, the reason hCG administration has been preferred to LH in clinical practice resides in the historic availability of commercial preparations, rather than systematic, evidence-based demonstrations.No clinical trials have yet compared LH to hCG in male HH.Only a single case has been reported, comparing a daily low hCG dose ( IU) to r-LH ( IU daily) in a man with HH after surgery for a pituitary adenoma ().In that patient, either hCG or r-LH restored eugonadism without exhibiting a substantial difference.A careful comparison of the in vivo action of LH and hCG in HH men is needed.In  healthy men with pituitary suppression from a GnRH antagonist, it was demonstrated that a daily LH dose of .IU restored testosterone levels to the normal range ().That trial found no differences between bolus and pulsatile administration, suggesting that the physiological pulsatile LH secretion pattern is not strictly needed to obtain Leydig cell stimulation ().These two examples indicate that low doses of LH could be sufficient to stimulate testicular androgen production to an extent similar to that obtained by apparently much higher doses of hCG ( IU weekly for hCG vs .IU weekly for LH), revealing that the dosage of LH necessary to stimulate physiological testosterone production might be much lower than expected.If so, this would clearly indicate a differential action of LH and hCG in vivo in human males.Clinical studies with LH in HH men are urgently needed to explore this finding.
In women, the main LH clinical application remains COS during ART.Several trials have evaluated different ART outcomes using LH instead of hCG to support follicular growth (Fig. ) ().Despite their large numbers, these studies showed high heterogeneity and low quality, hindering the development of standard protocols based on scientific evidence.Thus, the gonadotropin stimulation during COS, claimed to be "personalized" by ART physicians, remains a peculiar example of personalized medicine, in that personalization is not based on the selection of the appropriate stimulus according to objective criteria but, rather, on the "personal" beliefs of the prescriber.This challenge is further complicated by the increasing number of young infertile couples seeking ART and the corresponding cost of gonadotropins and procedures, which leaves the pharmacological approach widely driven by the industry.
In this confusing setting, several clinicians have proposed the addition of LH activity to FSH during COS.This relatively new combination has been widely debated in the reported studies, in particular regarding those with a poor response and women of advanced age (-).Available evidence does not clearly support the hypothesis of increased pregnancy rates using LH combined with FSH in unselected women (), and the number of reported meta-analyses almost outweigh the number of RCTs available on this topic, suggesting the difficulty in designing an objective meta-analysis to identify the best COS approach, distinguishing the actual benefit of one treatment compared with another ().Apart from the paucity of adequately powered studies and the extremely high heterogeneity of the treatments used, this topic has been weakened by the heterogeneity of the endpoints evaluated in RCTs ().The vast majority of RCTs and meta-analyses have focused on the ART clinical outcome.However, when the effectiveness of the addition of LH and/or hCG is the topic of investigation, the first endpoint to be evaluated must be the ovarian response (i.e., the first measurable parameter of gonadotropin action).Pregnancy and live birth rates, in contrast, are the final results of ART and influenced by an increasing number of factors (e.g., sperm contribution, endometrial receptivity and implantation, placenta).A recent meta-analysis combined the reported results, considering all gonadotropin combinations and all ART outcomes, demonstrating that FSH alone resulted in greater oocyte numbers compared with hMG or FSH plus LH ().The addition of LH activity is useful for reducing the amount of FSH needed and to improve oocyte quality but only when LH is used rather than hCG.This could be a further suggestion that by reducing the FSH doses used, the FSH activity in granulosa cells is shifted from the proapoptotic to the proliferative pathways, such as those activated by LH (, , ).Only five studies have directly compared LH to hCG; however, none clearly reported the hCG dosage combined with FSH, preventing any estimation of the real exposure of the ovary to LH/hCG in terms of the molar relationship between LH or hCG and the LHCGR.
With the limitation of the pharmacological, rather than the physiological, setting, these results suggest that it is difficult to establish the correct amount of LH to be used in clinical practice, which might be different depending on the clinical setting (HH or COS) and the patient's sex.In addition, considering the difficulty of measuring in vivo LH bioactivity using approved bioassays, the debate remains fully open.The correct appraisal of the clinical application of LH should probably combine in vitro and in vivo demonstrations.

Complications of LH and hCG therapy
In the evaluation of possible clinical applications of LH and hCG, adverse events must also be considered.The most relevant iatrogenic COS adverse event remains OHSS, which is the final result of an exaggerated ovarian response ().It rarely occurs when ART is applied to unselected women (incidence of % to %); however, its incidence increases when high-risk women are treated ().This risk is associated with a low body mass index, young age, high estradiol serum levels, high IL concentrations, high vascular EGF, and renin-angiotensin system activation, although specific predictive parameters are not yet available ().Moreover, either the elevated estradiol serum levels obtained at the day of ovulation or the large number of follicles developed during COS could predispose to the development of OHSS ().These OHSS-predisposing conditions could be related to gonadotropin action and should be carefully evaluated to predict the occurrence of adverse ART events.hCG, rather than LH, is generally used to trigger ovulation.In addition to differences at the molecular level, LH and hCG have different circulating half-lives ().The sustained luteotropic activity induced by hCG could lead to side effects through the release of vasoactive substances (e.g., vascular EGF) and prostaglandins acting directly on the ovarian follicles.These direct and indirect effects could result in OHSS ().Triggering ovulation with a GnRH agonist instead of a classical hCG bolus seemed to reduce the OHSS risk (, ).A recent meta-analysis reported a reduced OHSS rate with a GnRH agonist compared with hCG-triggered cycles, although no statistical significance was reached ().These findings have confirmed the lack of clear knowledge of OHSS pathophysiology.To enhance the knowledge of underlying pathophysiological mechanisms and to reduce the incidence of this adverse event, the two different clinical OHSS presentations should be considered separately ().First, early OHSS occurs within  days after hCG administration as a final, prolonged hCG effect on already stimulated ovaries.This OHSS clinical picture could probably benefit from replacement of the hCG trigger by a GnRH agonist.Late OHSS occurs .days after triggering, representing the ovarian response to the endogenous hCG increase after fertilization ().This late complication does not benefit from hCG substitution, and further research is needed to better understand this condition and its prevention. .
In addition to OHSS, other clinical COS complications should be considered.Among these, the most frequent adverse effect is cycle cancellation, which occurs in .% to .% of women of advanced age ().This event leads to important socioeconomic consequences owing to the need to repeat COS, further exposing women to the risk of OHSS and an increasing economic burden.The main reason for cycle cancellation remains an inadequate response to gonadotropin stimulation.This probably results from both the lack of standardization in COS protocols and the high heterogeneity of the clinical responses.The only chance to reduce the cycle cancellation rate is to develop truly, evidence-based personalization of COS schemes, which will require rigorous clinical studies.
In HH men, the only adverse event that must be considered during hCG/LH treatment is excessive serum testosterone levels, with possible negative effects on red blood cells, liver, and prostate gland.However, this effect is the result of androgen action on sensitive tissues rather than a direct consequence of hCG administration.It will be interesting to explore whether HH treatment by LH rather than hCG will reduce the rate of adverse events.Similar conclusions can be applied to the pharmacological treatment of cryptorchidism.Cryptorchidism is identified by the absence of one or both undescended testes to the scrotum and is linked to germ cell loss, infertility, and increased testicular cancer risk ().The descent of the testes can be induced by hCG administration or surgery.Treatment of cryptorchidism with hCG was associated with germ cell apoptosis and impaired reproductive function in the adult ().This effect is consistent with the cAMP/PKA-dependent proapoptotic potential of hCG and might plausibly be dampened by administering LH instead of CG.However, the use of LH as a treatment of cryptorchidism has never been reported.

Beyond Reproduction: Effects of hCG on Thyroid and Adrenal Glands
Thyroid hormones and TSH are involved in metabolism and development regulation, and increasing evidence has highlighted a role for these molecules even in reproduction (), revealing cross-talk between the gonadal and thyroid axes in vertebrates ().High structural and biochemical similarities between gonadotropins and TSH (), and their receptors, underlie such interactions.The binding affinity of LHCGR and TSH receptor (TSHR) for their respective ligands relies on amino acidic residue at positions - of the extracellular domains.These were considered to lead to chimeric receptors for hormone cross-interaction studies in the past ().hCG-TSHR chimeras revealed -fold cAMP increases in transfected HEK cells, and the effect was mimicked by high concentrations (. 3   to   ng/mL) () of hCG acting on wild-type TSHR in vitro, demonstrating cross-activation of the canonical cAMP/PKA pathway () and iodide uptake and triiodothyronine secretion in cultured human thyrocytes ().An in vitro comparison of LH and hCG using a CHO cell line permanently expressing TSHR cDNA (CHO-JP cells) revealed that LH is more potent than hCG in inducing an intracellular cAMP increase, and a parallel line analysis demonstrated the overall equivalency of . mM hCG equal to . mM LH equal to . 3  2 mM TSH ().A mutant hCG lacking the CTP fragment displayed similar potency to LH, suggesting that the C-terminal peptide might act as a protective factor to prevent hyperthyroidism due to hCG crossreactivity during pregnancy, when circulating concentrations of this hormone are extremely high.However, both LH and hCG are able to displace TSH bound to its receptor, thereby revealing an unexpected, ~-fold greater, thyrotropic activity in LH compared with hCG, equivalent to . and . mIU of TSH activity per . IU of LH and hCG, respectively ().In CHO-JP cells, enzymatic digestion of hCG resulted in deglycosylated and/or desialylated molecules with greater potency in activating cAMP compared with classical hCG (), suggestive of plausible evolutionary conservation of the TSH dependence of thyroid function during the early weeks of pregnancy.Similar results were obtained with hCG preparations from patients with trophoblastic disease.These studies support the concept of thyrotropic action of gonadotropins, especially hCG, which reaches relatively high serum levels during pregnancy or in patients affected by hCG-secreting tumors.It is well known that the increase in hCG levels during the th to th weeks of pregnancy matches the decrease in TSH levels and sustains continuous thyroid hormone production ().This likely depicts the dual function of hCG, consisting of negative feedback exerted through thyroid hormones at the pituitary and supporting thyroid function.These effects are emphasized during twin pregnancies, which features prolonged, higher hCG levels compared with single pregnancies and more frequently increased free T and suppressed TSH levels ().In contrast, insufficient thyroid stimulation could result in low thyroid hormone levels and could result in spontaneous abortion ().Taken together, the metabolic regulation by thyroid hormones might be, in part, dependent on hCG during pregnancy and might be essential to support fetal development.
Because TSHR and thyroid hormone receptors are expressed in endometrial cells, their ligands might be involved in the physiological regulation of this tissue (), although clear-cut evidence remains to be found.Given the relatively high potency of LH in inducing TSHR-mediated cAMP production in vitro (, ), a role for gonadotropins exerted at the level of the female reproductive system through the TSHR cannot be excluded.LH and TSH release by the pituitary gland seems to be synchronized, at least during the menstrual cycle, and is largely measurable as a serum concentration peak of both hormones ().Also, it is known that hypothyroidism before puberty leads to delayed sexual maturity, implying thyroid hormone action on gonadal function ().However, the promiscuity of endocrine signals was demonstrated even in simple vertebrates, such as fish (), suggesting that it is a conserved feature regulating certain physiological functions.It is not surprising that hypo-and hyperthyroidism are associated with menstrual disturbances, anovulation, and subfertility in females ().The cross-talk between thyroid and gonadal functions is far from being thoroughly elucidated.The relatively recent discovery of thyrostimulin (), a gonadotropin-like molecule acting on the TSHR and considered the most ancestral glycoprotein hormone, has provided information on ovarian functioning.The production of thyrostimulin by oocytes has been described, suggesting that this molecule, rather than TSH, might act as a paracrine factor in the ovary by binding TSHR expressed in granulosa cells (), where it would induce proliferative signals, simultaneously activating cAMP/ PKA, ERK/, and AKT pathways ().Although suggestive, these findings should be confirmed by clinical evidence, which is required to understand the whole picture of the thyroid-gonadal interaction.
Interactions between LH/hCG and the adrenal gland were described in healthy women during the menopausal transition, as an effect linked to increasing serum LH concentrations and, during the first trimester of pregnancy, to hCG production.The adrenal gland intriguingly shares a common developmental origin with gonadal cells ().It is characterized by different types of cells secreting mainly glucocorticoids, as well as androgens and a small amount of estrogens.During the menopausal transition, the lack of progesterone-negative feedback to the pituitary, occurring with the arrest of follicular maturation, results in increasing levels of LH molecules acting on LHCGR expressed in adrenal cortical cells ().The adrenal gland response consists of the synthesis of pregnenolone metabolites obtained via the D-steroidogenic pathway (), mainly dehydroepiandrosterone and androstenediol (), as well as cortisol ().Most importantly, chronically elevated LH levels are suspected to be linked to an increased risk of the development of adrenal tumors (), although further studies are required to elucidate the effect of LH on adrenal function.Long-term elevated LH levels induce Lhr expression in mouse adrenal glands, a first step required for initiating cortical tumor development (), and it is possible that such an effect also occurs in humans ().hCG action in the adrenal gland was discussed in the section "Pathophysiology of LH and hCG."In certain cases, increased hCG has been suspected to be linked to adrenal LHCGR expression during pregnancy, resulting in high cortisol levels and transitory Cushing syndrome ().However, hCG-stimulated synthesis of adrenal steroids is marginal in healthy women () and transient Cushing syndrome is a relatively rare event.

Conclusions
The evolution of different glycoprotein hormones and their receptors accompanied the separation of distinct endocrine axes to optimize endocrine and metabolic functions, which developed independently in vertebrates.Interspecies ligand-hormone binding is possible, depending on the phylogenetic distance of the species and suggesting evolutionary conservation of key amino acid residues fundamental for the preservation of biochemical properties of the proteins.Intraspecies interactions between endocrine axes are also possible, owing to the common developmental origin of the hormone target cells or as a method of optimizing certain metabolic functions.However, the complexity of the physiological mechanisms mediating placentation of certain mammals, such as equids and primates, required a more profound regulatory level, making the role of gonadotropins of pituitary and chorionic origin distinguishable.It is plausible that this complexity achieved its maximal expression in humans, with the largest number of genes encoding for LHb/CGb molecules likely evolving from a single ancestral gene.Although these hormones might exist in more than one isoform and glycosylation variant, each displaying specific biochemical properties, their physiological roles might be separated according to specific functions required for gamete maturation and pregnancy support (Table ).
LH production is maintained in both males and females during the fertile age, when their function depends on the specific enzymatic milieu of target cells.In women, the known androgenic role of LH is fulfilled by an overall flattened production of androstenedione by theca cells, as the major steroid serving as a substrate to be converted to the proliferative factor, estradiol, by granulosa cells under FSH stimulation.However, both progestational and proliferative LH-dependent effects have been shown to be relevant, specifically in granulosa cells, by in vitro experiments, revealing previously unknown regulatory molecular mechanisms, suggesting that the aforementioned metabolic functions are fundamental for proper oocyte maturation.These roles are exerted through a fine-tuned modulation of the balance between the steroidogenic cAMP/PKA pathway on the one hand and proliferative/antiapoptotic signals mediated through ERK/ and AKT pathway activation on the other, thus exhibiting the capability of biased signaling mediated under synergistic modulation exerted by FSH.Hence, female folliculogenesis is assisted by two LH and one FSH target cells and characterized by both LHCGR and FSHR expression "Clinical comparisons of LH and hCG in those with HH are needed to clarify many outstanding questions." as a key factor for sustaining reproductive function.This dual regulatory system might be involved in dampening signaling defects, resulting in overall mild pathological phenotypes, but not necessarily leading to failed pubertal development or complete infertility, such as in the case of PCOS.LH androgenizing function is maintained in the male counterpart of theca cells, the Leydig cell, where the steroidogenic pathway is driven toward testosterone synthesis as a major product required for male sexual development and reproduction.In this case, the lack of LH function leads to individuals with impaired sexual development, exacerbated by LHCGR-inactivating mutations.Defective hCG signaling is naturally linked to pregnancy defects, mainly miscarriage, owing to inappropriate progesterone synthesis.In contrast, excessive hCG signals might increase the risk of maternal, transitory Cushing syndrome or adrenal tumors.Trophoblast hCG might appear in women when the gonadal steroidogenic machinery is set to produce exclusively progesterone by luteinized ovarian cells, thus not requiring any discrimination between proliferative and steroidogenic signals.This was demonstrated by hCG treatment of granulosa cells in vitro, in which hCG was not able to reproduce LH-specific proliferative signals but exhibited a relatively high steroidogenic potential.In addition, LH and hCG treatment of mouse Leydig cells failed to demonstrate any qualitative differences, revealing the sex-specific nature of LH and hCG signaling.These data underline the significance of LH and hCG application to fertility treatments, especially in women, in whom they have been classically used as equivalents.This is an oversight caused by misinterpretation of the clinical data provided by studies performed in the ART context, in which the LH-and hCG-specific effects are masked by those that occur after the massive estrogen production induced to achieve multifollicular development, which does not normally occur in humans.Clinical comparisons of LH and hCG in those with HH is needed to clarify many outstanding questions.

Figure 1 .
Figure 1.Genetic, structural, and functional relationships between mammal LHb an CGb molecules.Mice have a unique Lh molecule

Figure 2 .
Figure2.Differential free LHb and hCGb subunit release by somatotrope GH3 cells.Both hormones have an asparagine (N) residue at position 13 of the amino acid chain (N13), which is glycosylated in the hCG molecule.N13 glycosylation prevents the disulfide-linked aggregation of b subunits and is involved in maintenance of the correct folding in the absence of the a subunit.This mechanism might underlie the secretion of free hCGb subunits occurring in pregnancy, when these peptides are produced in greater amounts than the a subunits, with free LHb aggregates remaining in the cytoplasm.Vertical line indicates conserved amino acid residues; two vertical dots, different amino acid residues sharing similar biochemical properties; and one dot, biochemically different amino acid residues.

Figure 3 .
Figure 3. Discrimination of LH-and hCG-mediated signaling stratified by the LHCGR hinge region.Both hormones bind the extracellular domain of the receptor but interact differently with the "Ushaped" portion of the hinge region.(Upper) Although hCG contacts the exon 10-encoded portion, LH spatial conformation leads to the interaction of the hormone with the sTyr331 residue.(Lower) These ligand-receptor interactions result in LHCGR conformational changes associated with hormone-specific intracellular signaling.Exon 10 deletion results in the shift of the sTyr residue impairing the interaction with LH, with a contact point of the "U-shaped" structure of the hinge region with hCG maintained.Thus, exon 10 deletion results in a truncated LHCGR unable to mediate proper LH signaling, albeit retaining both LH and hCG binding capability.

Figure 4 .
Figure 4. Comparison of LH-and hCG-mediated signaling in the ovary and testis.At the midantral

Table 1 .
Evolutionary and Genetic Differences Between LH and hCG

Table 2 .
Molecular Differences Between LH and hCG

Table 3 .
Effective Concentration for cAMP, pERK1/2, and pAKT Activation in Primary Mouse Leydig and Human

Table 4 .
Available Drugs With LH and/or hCG Activity According to Current Regulatory Agencies

Table 5 .
Physiological Differences Between LH and hCG