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Normal Spermatogenesis in a Man with Mutant Luteinizing Hormone

Caroline Achard, Ph.D., Carine Courtillot, M.D., Olivier Lahuna, Ph.D., Gri Mduri, M.D., Ph.D., Jean-Claude Soufir, M.D., Ph.D., Philippe Lire, Ph.D., Anne Bachelot, M.D., Ph.D., Hassani Benyounes, M.D., Michael Schumacher, Ph.D., Frdrique Kuttenn, M.D., Philippe Touraine, M.D., Ph.D., and Micheline Misrahi, M.D., Ph.D.

Sum m a r y
Men with mutations in LHB, the gene encoding the beta subunit of luteinizing hor mone (LHB), have azoospermia with absent or few fetal Leydig cells. We report a mutation in LHB in a man and his sister. The man presented with absence of viril ization, undetectable luteinizing hormone, and a low serum testosterone level. He had complete spermatogenesis with a normal sperm count. The mutant luteinizing hormone had a low level of partial activity in vitro. We concluded that the residual luteinizing hormone activity, resulting in the expression of steroidogenic enzymes in few mature Leydig cells producing small amounts of intratesticular testosterone (20.2 ng per gram), was sufficient for complete and quantitatively normal spermato genesis.

From INSERM Unit 854, University Paris South 11 (C.A., O.L., M.M.); and the Laboratories of Molecular Genetics, Pharmacology, and Hormonology (G.M., M.M.) and Biological Andrology (J.-C.S.), Assistance PubliqueHpitaux de Paris; and INSERM Unit 788 (P.L., M.S.) all at Bictre Hospital, Le Kremlin Bictre, France; Universit Paris VI, Department of Endocrinology and Reproductive Medicine, PitiSalptrire Hospital, and Centre des Maladies Endocriniennes Rares de la Croissance (C.C., A.B., F.K., P.T.) and University Paris Descartes (O.L., J.-C.S., M.M.) all in Paris; and Abdou Hay Al Mahatta, Oujda, Morocco (H.B.). Address reprint requests to Dr. Misrahi at INSERM Unit 854, Laboratory of Molecular Genetics, Pharmacology, and Hormonology, Assistance PubliqueHpitaux de Paris, Bictre Hospital, 94275 Le Kremlin Bictre, France, or at micheline. misrahi@bct.aphp.fr. Drs. Achard, Courtillot, and Lahuna contributed equally to this article, as did Drs. Touraine and Misrahi. N Engl J Med 2009;361:1856-63.
Copyright 2009 Massachusetts Medical Society.

utations that abolish the activity of luteinizing hormone are rare; they have been reported in five men and one woman.1-5 The phe notypes of these persons suggest that luteinizing hormone is not required for male sexual differentiation but is critical to the proliferation and function of Leydig cells and to the induction of puberty. Infertility and very low levels of sper matogenesis persist in the affected men, despite long-term exposure to human cho rionic gonadotropin, suggesting that the absence of perinatal exposure to luteiniz ing hormone alters Leydig cells proliferation and maturation, impairing the onset of normal spermatogenesis, which is thought to be critically dependent on a high level of intra testicular testosterone.6-9 We report a case of familial hypogonadotropic hypogonadism involving a partial loss of luteinizing hormone function. The proband had an undetectable circulating luteinizing hormone level and a low serum testosterone level. He had a markedly small population of mature Leydig cells expressing the steroidogenic enzymes necessary for androgen synthesis and producing low levels of intratesticular testos terone. He had a mutant form of luteinizing hormone beta that showed low-level residual function in vitro, indicating the presence of low levels of luteinizing hor mone activity from birth to adulthood, which permitted the maturation and func tion of a small number of Leydig cells. This was nevertheless sufficient to trigger and maintain complete and quantitatively normal spermatogenesis.

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brief report

C a se R ep or t
This study was approved by the review boards of the institutions involved, and written informed consent was obtained from the patient and his family members. The 43-year-old proband (Sub ject IV-3, with a 46,XY karyotype) (Fig. 1) had been born in Morocco to consanguineous parents and was previously treated there for hypogonad ism at 28 years of age. Treatment with intramus cular testosterone resulted in masculinization and penile growth. The treatment was irregular be cause of poor adherence; it had been discontin ued for 3 months before the patient was referred to our department. Examination showed that the patient was virilized (185 cm in height, 75 kg in weight) with normal masculine features: pubic hair of Tanner stage 5, axillary hair of Tanner stage 3, penile length of 12 cm, normal testicular volume, and absence of gynecomastia. Initial and subsequent laboratory tests (Table 1) showed un detectable luteinizing hormone levels (i.e., <0.05 IU per liter), high follicle-stimulating hormone and anti-Mllerian hormone levels, normal in hibin B levels, and very low serum testosterone levels. Magnetic resonance imaging of the brain and pituitary was normal. Ultrasonography re vealed bilateral testicular microlithiasis and testis volume in the lower normal range (right testis, 14.5 cm3; left testis, 12.0 cm3). Hypogonadism due to isolated deficiency of luteinizing hormone beta was suspected. Surpris ingly, several spermiograms performed over a 10-month period consistently revealed normal numbers of spermatozoa (76.4106 to 127.0106 spermatozoa per milliliter) and qualitatively normal-to-subnormal spermatogenesis (5 to 23% of sperm with normal morphology and 25 to 35% with motility), with low semen volume (0.8 cm3).10 Testicular biopsy showed heterogeneous semi niferous tubules separated by fibrous tissue (Fig. 2A), containing few mature, vacuolated Leydig cells (Fig. 2B and 2C). Half the tubuli were hyalinized or hypoplastic, with a thickened lami na, immature Sertoli cells, and spermatogonia. The other half were composed of mature Sertoli cells and germ cells at all stages of differentia tion (Fig. 2A, 2B, and 2C). Immunohistochemical analysis showed that the small number of typical mature Leydig cells expressed enzymes necessary for androgen syn thesis: cytochrome P-450 (CYP) 17-hydroxylase

A
I II III
* 1 1 2 2 3 4 3 1 1 1 * 4 2 2 3 2 * 5 * 6 4

IV V

B LHB Gene
F E1 I1 E2 I2 E3 R

CCA TGG TGC CAC CCC ATC AAT GCC ATC Wild-Type P W C H P I N A I LHB CCA TGG TGC - AAT GCC ATC P W C N A I

Mutant LHB

Figure 1. Pedigree of the Patient with Hypogonadism and LHB Mutation. RETAKE 1st AUTHOR: Achard ICM Panel A shows the pedigree of the proband (Subject IV-3, 2nd FIGURE: 1 of 3 REG F 3rd arrow). Squares indicate men, circles women, and symCASE Revised bols with a slash persons who have died. The double Line 4-C SIZE line EMail indicates consanguinity. For the family members ARTIST: ts 16p6 H/T H/T whoEnon underwent genetic testing (denoted with an asterCombo isk), solid symbols indicate homozygosity for the LHB AUTHOR, PLEASE NOTE: mutation, and half-solid symbols heterozygosity. Panel Figure has been redrawn and type has been reset. Please check region carefully. B shows the complete coding of the LHB gene, including exons E1 through E3 and introns I1 and I2. JOB: 35119 ISSUE: 11-05-08 The nine-base deletion in exon E2 results in the deletion of three amino acids from the protein. (For wildtype and mutant LHB, the three-base codons are listed above the corresponding amino acid, represented by its single-letter symbol.) Forward (F) and reverse (R) primers were used in the polymerase-chain-reaction restriction-fragmentlength polymorphism analysis (see Fig. S1B in the Supplementary Appendix), as indicated.

(Fig. 2D), 3-hydroxysteroid dehydrogenase (Fig. 2F), and CYP cholesterol-side-chain cleavage en zyme (not shown). We also detected fibroblastlike fetal Leydig precursors, located outside the tubular basal lamina, expressing 3-hydroxyste roid dehydrogenase (Fig. 2G) but devoid of CYP 17-hydroxylase (not shown).9,11 There were mark edly fewer Leydig cells in the patients testistissue sample than in a sample from an agematched control (Fig. 2E). The patients Sertoli cells expressed anti-Mllerian hormone (Fig. 2H), the persistence of which indicates low levels of intratesticular testosterone,12 whereas control
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LH Baseline yr 43 46 32 40 71 0.87.6 1.111.6 3.346.4 3.014.4 4.528.8 0.711.1 3.3010.00 0.202.90 24.5 42.3 <10 <35 3050 for follicular phase, 150230 for luteal phase 1060 60.0330.0 3.05.4 <0.70 2.4 5.4 4.00 4.5 4.6 66 ND 53.3 13 ND ND 20.7 29.8 0.48 0.62 <10 29 IU/liter ng/ml g/liter pg/ml 71.6 Peak Baseline Peak ng/ml 9.3 IU/liter 1.28
The

Table 1. Serum Hormone Levels in the Proband with LHB Mutation, His Affected Sister, and Members of Their Family.* FSH Testosterone Androstenedione 17-Estradiol Estrone Inhibin B AMH Subunit

Subject No.

Age at Sex Baseline

IV-3 (proband)

IV-5 (affected sister)

IV-4 (unaffected)

IV-2 (unaffected)

III-2 (unaffected)

Normal range

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* The peak value was the maximum level measured within 90 minutes after the intravenous administration of 100 g of gonadotropin-releasing hormone. Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by means of an immunometric chemiluminescence assay (Siemens). The intraassay coefficient of variation was between 3.40% and 13.10% for LH (the latter value, at levels 0.15 IU per liter) and between 2.9% and 3.4% for FSH. The interassay coefficient of variation was between 6.2% and 23.9% for LH (the latter value, at levels 0.15 IU per liter) and between 4.1% and 4.8% for FSH. The sensitivity of the LH assay was 0.05 IU per liter. The testosterone level was determined with the use of a radioimmunoassay (Orion Diagnostica). The intraassay and interassay coefficients of variation were between 3.8% and 7.5% and between 4.8% and 7.0%, respectively. The sensitivity of the testosterone assay was 0.029 ng per milliliter (0.100 nmol per liter) (limit of measurement, 0.140 to 14.400 [0.5 to 50.0]). Serum levels of androstenedione, estradiol, estrone, inhibin B, anti-Mllerian hormone (AMH), and the subunit were assayed as described in the Supplementary Appendix. To convert values for testosterone to nanomoles per liter, multiply by 3.467. To convert values for androstenedione to nanomoles per liter, multiply by 3.492. To convert values for 17-estradiol to picomoles per liter, multiply by 3.671. To convert values for estrone to picomoles per liter, multiply by 3.699. ND denotes not detectable. Data for the proband, Subject IV-3, are from the initial laboratory assays performed during the first consultation, 3 months after the last testosterone injection. Subject IV-5 is the probands sister, who had amenorrhea and infertility. Data for Subject IV-4, another sister of the proband, are from tests performed on day 25 of her menstrual cycle. The pedigree is shown in Figure 1. Intratesticular hormone levels in the proband are shown in Table S1 of the Supplementary Appendix. The mean (SD) value for castrates in the testosterone assay is 0.250.03 ng per milliliter.

brief report

cells had an absence of anti-Mllerian hormone (Fig. 2I). The expression patterns of the androgen receptor (Fig. 2J and 2K) and of both markers of germ-cell-maturation histone H1 (Fig. 2L and 2M) and proacrosin (Fig. 2N and 2O) were similar in samples from the patients testis and the control testis. Testosterone measurements were obtained with the use of gas chromatographymass spec trometry.13 The level of intratesticular testoster one in the patients biopsy sample (20.2 ng per gram of tissue) was approximately one eighteenth that of a specimen from an age-matched control (356.6 ng per gram of tissue); the control value matches values in previous reports.14 Similarly, the level of intratesticular 5-dihydrotestosterone was much less in the patients sample (0.59 ng per gram of tissue) than in the control sample (12.5 ng per gram of tissue) (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). The serum testos terone level was 0.4 ng per milliliter (1.4 nmol per liter), confirming the immunoassay results. The probands siblings (Fig. 1A) underwent normal, spontaneous puberty. One sister, Subject IV-5 (born in 1961), had menarche at 14 years of age and subsequently had oligomenorrhea and secondary amenorrhea. Repeated ultrasonogra phy in the sister, for evaluation of infertility, at 30 years of age revealed bilateral ovarian macro cysts. Recent assays showed undetectable luteiniz ing hormone and low estradiol and high folliclestimulating hormone levels (Table 1). A brother, Subject IV-2 (born in 1967), was the father of two children, and another sister, Subject IV-4 (born in 1975), had normal hormonal levels (Table 1). Yet another sister, Subject IV-1 (born in 1957), was the mother of two children; she and the other brother, Subject IV-6 (born in 1977), were unavailable for study.

Functional Analysis of Mutant Luteinizing Hormone Beta

Coding sequences of human LHA (encoding the alpha subunit of luteinizing hormone) and LHB were inserted into the pSG5 vector (Stratagene). We then introduced the deletion carried by the proband into LHB to create a mutant luteinizing hormone beta construct; we also introduced the V5 epitope into the C-terminal of the luteinizing hormone alpha subunit to make the construct -V5. Western blot analyses were performed with the use of an antihuman chorionic gonadotro pin antibody (ab14301, Abcam), which crossreacts with the beta subunit of human luteinizing hormone; the samples blotted were cell lysates and concentrated medium of human embryonic kidney (HEK) 293T cells transfected with the use of a transfection reagent (SuperFect, Qiagen). For coimmunoprecipitation analysis, cell lysates from transfected COS-7 cells (derived from fibroblasts from kidneys of African green monkeys) were in cubated with anti-V5 antibody (Invitrogen), and immunoprecipitates were analyzed by means of Western blotting. The activity of recombinant se creted mutant luteinizing hormone relative to that of wild-type luteinizing hormone was deter mined by assessing cyclic AMP accumulation in HEK 293T cells transiently expressing the human luteinizing hormone receptor. The level of wildtype luteinizing hormone in the culture medium was determined by means of immunofluoromet ric assay (LHsp enzyme-linked immunosorbent assay, Biosource). Details are given in the Supple mentary Appendix.

R e sult s
Sequence analysis of LHB from the proband (Fig. S1A in the Supplementary Appendix) revealed a homozygous nine-base deletion in exon 2, pre dicted to result in a deletion of amino acids 10 to 12 (histidineprolineisoleucine) in the mutant luteinizing hormone beta (Fig. 1B). Proline and the hydrophobic residues (isoleucine or valine) are highly conserved in mammalian LHB genes. The affected sister, Subject IV-5, was also homozy gous for the deletion; the mother (Subject III-2) and asymptomatic siblings (Subjects IV-2 and IV-4) were heterozygous for the deletion (Fig. S1B in the Supplementary Appendix). Both the mutant and wild-type luteinizing hor mone beta (both approximately 15 kD in size)16
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Me thods
DNA Sequencing

We sequenced the three exons of LHB, as described in the Supplementary Appendix.

Immunohistochemical Analysis

Immunohistochemical evaluation was performed with the use of a commercial kit (LSAB+ system with 3-amino-9-ethylcarbazole, Dako), as described previously.15

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Figure 2. Histologic and Immunocytochemical Studies of the Patients Testis. Panel A shows seminiferous tubules separated by a fibrous interstitium. In Panel B, a few vacuolated Leydig cells are visible (arrow). Panel C shows all stages of germ-cell differentiation, from spermatogonia (arrow) to spermatozoids RETAKE 1st Achard/Misrahi AUTHOR ICMmature (arrowhead); there is also an isolated, Leydig cell in the interstitium (asterisk). (In Panels A, B, and C, stain2nd REG F FIGURE 2a-o D, only a few interstitial androgen-producing ing was performed with hematoxylin and eosin.) In Panel cells positive 3rd CASE TITLE for cytochrome P-450 (CYP) 17-hydroxylase are visible (arrows), whereas such cells are abundant in a sample from Revised EMail 4-C Line an age-matched control (Panel E). We detected 3-hydroxysteroid dehydrogenase expression in both the few interSIZE Enon mst H/T H/T (Panel stitial, mature Leydig cells also expressing ARTIST: CYP 17-hydroxylase F) and in the fibroblast-like precursors, adjaFILL 33p9 Combo cent to the tubular basement membrane and lacking CYP 17-hydroxylase (Panel G, arrows). Panel H shows Sertoli AUTHOR, PLEASE NOTE: cells strongly expressing anti-Mllerian hormone, whereas no such expression is observed in the control sample Figure has been redrawn and type has been reset. (Panel I). (In Panels D through I, staining was performed with hematoxylin.) The expression pattern of the androgen Please check carefully. receptor was identical in interstitial and intratubular cells from the patient (Panel J) and from the control (Panel K). A similar pattern of expression of histone a marker of germ-cell maturation, 36119 11-5-09was observed in maturing germ JOB: H1, ISSUE: cells obtained from the patient (Panel L) and the control (Panel M). Proacrosin was detected in spermatids and spermatozoids from both the patient (Panel N, arrow) and the control (Panel O, arrow), indicating their advanced degree of maturation. Panels D, E, H, I, J, K, L, and M are at the same magnification; Panel A is at a slightly lower magnification; Panels B, G, N, and O, twice the magnification; and Panels C and F, four times the magnification.

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brief report

B
Immunoprecipitation Cell Lysate Culture Medium
ild M type ut an LH M t LH bet a oc k- bet tra a W ns ild fe c M type ted ut an LH M t LH bet a oc k- bet tra a ns fe ct ed

C
3000 2500 Wild-type LH Mutant LH Mock-transfected

Cell Lysate Control Anti-V5 Antibody Cyclic AMP (nmol/liter)

ild M type ut an LH t L be H ta be W ta ild -ty pe W LH ild -ty be M pe ta ut an LH t L be H ta be ta

2000 1500 150 100 50 0

16 50 1 2 3 4 5 6

LH beta -actin

16 50 22 1 2 3 4 5

kD

kD

LH beta -actin -V5

lit er

lit er

g /

g /

40

60

Stimulant Dose of LH

Figure 3. Production and Heterodimerization in Mutant and Wild-Type Luteinizing Hormone (LH) Beta and Bioactivity of Mutant 1st RETAKE AUTHOR: Achard and Wild-Type LH. ICM FIGURE: 3 of 3 beta subunit in transfected 2nd F of Panel A shows low intracellular and secretedREG levels mutant LH cells. Western blot analysis was performed 3rd on cell lysate (lanes 1 through 3) and cultureCASE medium (lanes 4 to 6) of human embryonic kidney (HEK) 293T cells: cells producing the Revised 4-C LH alpha subunit and the wild-type LH beta subunit (lanes 1 and 4), Line cells producing theSIZE LH alpha subunit and the mutant LH beta subEMail ARTIST: ts H/T H/T unit (lanes 2 and 5), and mock-transfected cells (lanes 3 and 6). An anti-actin antibody 36p6 was used as a loading control (lanes 1 through 3). Enon Combo Identical volumes of culture medium, concentrated by a factor of about 20 for wild-type LH beta (lane 4) and about 400 for mutant LH AUTHOR, PLEASE NOTE: beta (lane 5), were loaded. Wild-type LH beta and mutant LH beta were immunodetected with an antihuman chorionic gonadotropin Figure has been redrawn and type has been reset. antibody (Abcam). Panel B shows low levels of dimerizationPlease of thecheck alpha subunit carefully. and mutant beta subunit of LH. Coimmunoprecipitation experiments were performed on cell lysates from COS-7 cells producing the -V5 construct and either wild-type or mutant LH beta. Immunoprecipitation was performed withJOB: the use of anti-V5 antibody (lanes 4 and 5) or11-05-08 with a nonimmune immunoglobulin as a control 36119 ISSUE: (lane 3). This was followed by immunodetection of wild-type LH beta and mutant LH beta, with the use of a polyclonal antihuman chorionic gonadotropin antibody (Abcam), or of the -V5 construct, with an anti-V5 antibody. An anti-actin antibody was used as a loading control (lanes 1 and 2). Panel C shows markedly lower levels of mutant LH bioactivity in HEK 293 cells expressing the human LH receptor. The secreted levels of wild-type LH were quantified by means of immunofluorometric assay and used to generate a doseresponse curve (see Fig. S2 in the Supplementary Appendix). Comparative quantification of secretion of wild-type and mutant LH beta was carried out through Western blot analysis (Panel A, reflecting one representative experiment). HEK 293 cells expressing the human LH receptor were stimulated with concentrated culture medium containing a similar amount of either wild-type LH or mutant LH beta. Concentrated culture medium from mock-transfected cells were used as a negative control. The mean results are shown for three independent experiments using each of three doses of mutant or wild-type LH to stimulate cyclic AMP production. I bars indicate standard deviations.

were synthesized in HEK 293T cells (Fig. 3A). However, we observed less mutant protein than wild-type protein in cell lysates (mean [SD] ratio of wild-type:mutant, 2.30.8, from three inde pendent experiments), suggesting that the dele tion may result in decreased stability of the mutant protein or affect the stability or transla tional efficiency of its corresponding messenger RNA. In concentrated culture medium, the level of wild-type protein (expressed as the mean [SD] of three independent experiments) was 67.98.6 times that of the mutant protein (Fig. 3A). After normalization of this ratio on the basis of the intracellular expression ratio, we calculated that the secretion level (expressed as the mean [SD] of three independent experiments) associated

with wild-type luteinizing hormone was 32.811.7 (mean [SD] of three independent experiments) times that associated with the mutant luteiniz ing hormone beta, when coexpressed with the alpha subunit in HEK 293T cells. Coimmunoprecipitation studies with COS-7 cells cotransfected with expression vectors con taining the -V5 construct and the wild-type or mutant luteinizing hormone beta subunit were performed to determine whether the defect in secretion of mutant luteinizing hormone beta re sulted from defective subunit heterodimerization. Immunoprecipitates from cell lysates incubated with anti-V5 antibody, as previously described,2 contained both subunits, indicating the occur rence of heterodimerization of the alpha and beta
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g /

lit er

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subunits (Fig. 3B). However, immunoprecipitates contained less mutant luteinizing hormone beta than the wild type (ratios in two independent experiments, 1:8 to 1:10). Cyclic AMP accumulation was markedly de pressed in association with the mutant luteiniz ing hormone as compared with the wild-type hormone (Fig. 3C). Assuming that most of the secreted mutant luteinizing hormone is dimerized, as is wild-type luteinizing hormone, the mean (SD) residual function (measured in three inde pendent experiments) of the mutant hormone was 0.730.32% of that of the wild-type hormone.

Discussion
The patient described here had an absence of spontaneous virilization, very low serum testos terone levels, and minimal luteinizing hormone activity but complete, quantitatively normal spermatogenesis. In humans, there are three waves of Leydigcell growth: the first, during the antenatal period, when growth is dependent on human chorionic gonadotropin; and the second and third, during the perinatal period and puberty, respectively, when growth is strictly under the control of luteinizing hormone.9-11 Male infants display transient postnatal activation of the gonadotro pin-releasing hormone pulse generator, inducing a surge in follicle-stimulating hormone, luteiniz ing hormone, and testosterone that is correlated with normal adult spermatogenesis and fertility.17 Partial stimulation of Leydig-cell proliferation, maturation, and function by luteinizing hormone may have occurred in the testes of our patient, both perinatally and during puberty, to induce spermatogenesis. The mutant luteinizing hormone in our patient is known to retain partial activity, for two reasons. First, his testicular biopsy speci men contained mature Leydig cells expressing the steroidogenic enzymes CYP cholesterol-sidechain cleavage enzyme and CYP 17-hydroxylase, which are required for testosterone synthesis the expression of these enzymes is strictly de pendent on luteinizing hormone.9,11 Second, the intratesticular testosterone level in the patient (20.2 ng per gram) greatly exceeded (by a factor of 40) the serum testosterone level, a discrepancy that is consistent with testosterone production within the testes. The low production of intratesticular testos
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terone and consequent weak testosterone secre tion into the serum (Table 1, and Table S1 in the Supplementary Appendix) were insufficient to in duce virilization but were nevertheless capable, in cooperation with a functional follicle-stimulat ing hormonegonadal axis, of exerting a local paracrine effect on contiguous Sertoli cells and inducing the development and maturation of seminiferous tubules that are required for com plete spermatogenesis.18 Our findings support the recent proposal that congenital hypogonadotro pic hypogonadism be managed both neonatally and at puberty through the administration of exogenous gonadotropins mimicking the physi ologic gonadotropin surges.17,19 As has been de scribed in men with other LHB mutations, our patient had inappropriately normal levels of in hibin B, given his high plasma level of folliclestimulating hormone.2,3 This may have been due to the insufficient development of the folliclestimulating hormoneinhibin feedback loop.20 The homozygous LHB deletion of the patient and two of his heterozygous, unaffected fam ily members was found to occur near a disul fide bridge forming the cystine-knot folding motif. This region may tolerate conformational changes.16 In male mice deficient in the luteinizing hor mone receptor, spermatogenesis proceeds until the elongated spermatid stage, and the mice are infertile.21 However, major differences in the hor monal regulation of spermatogenesis are now known to exist between primates and rodents. Thus, to obtain complementary information on the maintenance of spermatogenesis in normal men undergoing gonadotropin suppression, stud ies have been performed in men with the use of hormonal contraception protocols. On adminis tration of testosterone, both luteinizing hormone and follicle-stimulating hormone are decreased to minimal levels, suppressing intratesticular tes tosterone production in these men.22,23 Intrates ticular testosterone levels similar to the serum levels (3.81.3 ng per milliliter [13.24.5 nmol per liter])24 cannot maintain quantitatively normal sperm production. However, low intratesticular testosterone levels have been found to be suffi cient for the maintenance of low levels of sper matogenesis in some men with previously nor mal testicular development and normal sperm maturation.22 Currently, the minimal intratesticu lar testosterone level required to trigger and main

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brief report

tain normal sperm production in men is not known.6,22 This study goes against current wisdom in that it shows that complete and quantitatively normal spermatogenesis may be triggered and main tained by low levels of luteinizing hormone activity postnatally and at puberty. A better un derstanding of the intratesticular hormonal micro environment required throughout life (and espe cially during the perinatal and peripubertal periods) for adult spermatogenesis could result in better strategies to treat certain patients with infertility and to develop hormonal contraception methods for men.22,23
References
1. Weiss J, Axelrod L, Whitcomb RW,

Supported by grants from INSERM, the Socit Franaise dEndocrinologie, Assistance PubliqueHpitaux de Paris, and the French Ministry of Health (DHOS). No potential conflict of interest relevant to this article was reported. We thank G. Schaison, P. Roger, N. Lahlou, and E. Pussard for helpful discussions; N. Josso (INSERM Unit 293, Montrouge, France) and D. Escalier (Centre Hospitalier Universitaire Bictre, Le Kremlin Bictre, France) for kindly providing the anti-Mlle rian hormone and anti-proacrosin antibodies, respectively; Prof. N. Suganuma (Nagoya University School of Medicine, Handa, Japan) for kindly providing the pM2 and pM2 vectors; S. Maillet for help with sequencing analyses and polymerase-chain-reac tionrestriction-fragmentlength polymorphism experiments; C. Coussieu, S. Brailly, and N. Lahlou for performing hormonal assays and S. Bart for performing the testicular biopsy; and A. Pianos, B. Eychenne, and A. Cambourg for technical assistance with steroid determinations by means of gas chromatography mass spectrometry.

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