Inactivating Mutations of Luteinizing Hormone -Subunit or Luteinizing Hormone Receptor Cause Oligo-Amenorrhea and Infertility in Women

Women harbouring inactivating mutations in luteinizing hormone (LH) beta subunit ( LHB ) or LH receptor ( LHCGR ) genes have similar clinical manifestations characterized by female external genitalia, spontaneous breast and pubic hair development at puberty, and normal or late menarche followed by oligo-amenorrhea and infertility. Oestradiol and progesterone levels are normal for the early to midfollicular phase, but do not reach ovulatory or luteal phase levels, confirming lack of ovulation. Notably, serum LH levels are low in patients with LHB mutations and high in those with LHCGR mutations, whereas follicle-stimulating hormone levels are normal or only slightly increased. Pelvic ultrasound has demonstrated a small or normal uterus and normal or enlarged ovaries with cysts. Women with LHB mutations may be treated with hCG (human chorionic gonadotropin) or LH, whereas those with mutations in LHCGR are resistant. Lhb and Lhcgr knockout female mice are close phenocopies of the respective human mutations, and confirm that early follicular development, low levels of oestrogen production and theca cell development are independent of LH action, which Received: May 8, 2008 Accepted after revision: September 9, 2008 Published online: January 8, 2009 HORMONE RESEARCH Dr. Ivo Jorge Arnhold, Laboratório de Hormônios e Genética Molecular – LIM/42 Disciplina de Endocrinologia, Departamento de Clínica Médica, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Av. Enéas de Carvalho Aguiar 155 – PAMB – 2o andar – Bloco 6, São Paulo, SP, CEP 05403–900 (Brasil) Tel. +55 11 3069 7512, Fax +55 11 3069 7519, E-Mail [email protected] © 2009 S. Karger AG, Basel 0301–0163/09/0712–0075$26.00/0 Accessible online at: www.karger.com/hre D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /7 /2 01 7 2: 50 :0 5 A M Arnhold /Lofrano-Porto /Latronico Horm Res 2009;71:75–82 76 The human LH/CG receptor gene ( LHCGR ), located at chromosome 2p21, is composed of 11 exons. The largest exon, 11, codifies the entire transmembrane domain. Naturally occurring inactivating mutations in the LHB and LHCGR genes are very rare, especially in females, but constitute a model providing insight into their physiological role in human reproduction. The first woman with a mutation in the LH -subunit was described only recently [3] , where the phenotype resembled that of previously reported women with mutations in the LH receptor ( table 1 ) [4, 5] . Mutations in the genes that encode the gonadotropin subunits seem to be even more uncommon than those affecting the gonadotropin receptors. All female patients with LHB or LHCGR gene mutations reported to date are sisters of 46,XY probands with the corresponding mutations in LH or its receptor. However, it is important to be able to recognize this phenotype in the absence of an affected male relative. Inactivating LH  -Subunit Mutations Inactivating LH -Subunit Mutations in 46,XY Subjects To date, at least 5 men with inactivating LHB mutations from 4 different families have been reported [3, 6– 8] . The phenotype included male external genitalia at birth, but absent pubertal development and severe testosterone deficiency and azoospermia during adulthood. The testicular histological hallmarks of selective LH deficiency in these men were absent or hypoplastic Leydig cells and spermatogenic arrest. Notably, the case with a Q54R mutation in LHB had high immunoreactive LH levels measured by radioimmunoassay, but low LH receptor-binding properties on radioreceptor assay [6] . Lofrano-Porto et al. [3] have recently identified 2 men homozygous for a mutation in the 5 splice-donor site of intron 2, IVS2 + 1G ] C, resulting in an abnormal mRNA incorporating the 236 nucleotides of intron 2 of LHB . Interestingly, the administration of chronic exogenous testosterone to these affected men resulted in a significant increase in testicular volume. This effect was attributed to the synergistic actions of high endogenous FSH levels and increasing levels of circulating testosterone, which may have contributed to the late proliferation of Sertoli cells and testicular growth [3] . Inactivating LH -Mutations in 46,XX Subjects The sister of these 2 men is the sole woman described so far who harbours an inactivating mutation in the LH -subunit gene [3] . She had normal pubertal development and menarche at the age of 13 years, followed by oligomenorrhea for 14 years of her life. At the age of 29, her breasts ( fig. 1 ) and uterus were completely developed (uterine volume: 50 ml; normal range, 30–90 ml). Ovaries were of normal or slightly enlarged size (7 and 10 ml, normal 3–9 ml; fig. 2 ) and had a multicystic appearance, a finding consistent with the growth of multiple small follicles induced by FSH. Serum LH concentrations were undetectable both at baseline and after stimulation with Table 1. Characteristic features of women (46,XX) with inactivating mutations in LH -subunit or LH receptor External genitalia female Pubic hair and breast development normal Menstrual cycles normal to late menarche followed by oligo-amenorrhea Fertility absence of ovulation (LHB mutations may respond to hCG or LH) LH levels low in LHB and high in LHCGR mutations FSH levels normal to slightly high Estradiol and progesterone levels low to normal for midfollicular phase Pelvic ultrasound small to normal uterus, normal to enlarged ovaries with cysts Bone mineral density low to normal Fig. 1. Woman with LH -subunit mutation at 29 years of age. Complete breast development attained at puberty without hormonal replacement, revealing sufficient endogenous oestrogen secretion despite absence of LH. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /7 /2 01 7 2: 50 :0 5 A M LH and LH Receptor Mutations in Women Horm Res 2009;71:75–82 77 gonadotropin-releasing hormone, whereas FSH concentrations (4.8 IU/l) were normal. Repeated measurements of serum oestradiol (29–44 pg/ml) and progesterone (0.44 ng/ml) showed levels within the low-to-normal range for the follicular phase. Serum inhibin B level was high, further strong evidence for the presence of healthy early antral follicles in her ovaries. This Brazilian woman was found to harbour the same homozygous mutation in a non-coding intronic sequence affecting a 5́ splice-donor site of the LHB gene (IVS2 + 1G ] C). A G ] C substitution at position +1 of intron 2 of the LHB gene severely disrupted the splicing of the LHB mRNA, resulting in an exon 3 frameshift, consequently generating an aberrant transcript [3] ( fig. 3 ). Following supplementation with oestrogen, a dominant follicle of 18 mm was evident on ultrasonography, but ovulation remained impaired and no corpus luteum was identified. Interestingly, after 10 days of combined oestrogen-progestagen treatment, a functional follicular cyst measuring 44 mm in diameter was formed. Taken together, these findings corroborate the knowledge that female pubertal development may occur normally in the absence of LH action, reinforcing the main actions of LH on the induction of ovulation and corpus luteum formation. Furthermore, the formation of a dominant follicle after supplementation with oestrogen only may indicate that LH activity is not necessary until the early preovulatory stage of follicular development. This finding suggests that oestrogen or oestrogen-related factors were sufficient to induce dominant follicular growth up, until this stage, in the presence of preserved FSH secretion and a normal ovarian environment, as observed in the LH-deficient woman [3] . Inactivating LH Receptor Mutations Inactivating LH Receptor Mutations in 46,XY Subjects In genetic males, severe homozygous inactivating mutations of the LH receptor (LHCGR) cause 46,XY disorders of sex development, previously referred to as male pseudohermaphroditism [4, 9, 10] , whereas milder mutations result in hypospadias and/or micropenis and hypogonadism [4, 11–13] . In the absence of androgen action during human fetal development, the external genitalia remain female, irrespective of chromosomal or gonadal sex. hCG and LH bind to the same LHCGR at testicular Leydig cells. During the first trimester of gestation, hCG, by stimulating the LHCGR, is fundamental for secretion of testosterone and development of the male external and internal genitalia. Most inactivating mutations of LHCGR result in reduced or absent testosterone secretion and consequent impairment of masculinization of the external genitalia [14] . Interestingly, a deletion of exon 10 of LHCGR has been described, resulting in a phenotype of hypogonadism in a man with normal male external genitalia. In vitro, the LHCGR with this deletion responded normally to hCG, but not to LH, explaining Fig. 2. Enlarged ovary with large cysts in a woman with LH subunit mutation. mRNA