normal spermatozoa

7/30/2019 Normal Spermatozoa

1/8

brief report

T he n e w e n g l a n d j o u r n a l o fmedicine

n engl j med 361;19 nejm.org november 5, 20091856

Normal Spermatogenesis in a Man

with Mutant Luteinizing HormoneCaroline 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.

From INSERM Unit 854, University ParisSouth 11 (C.A., O.L., M.M.); and the Lab-oratories of Molecular Genetics, Phar-macology, and Hormonology (G.M.,M.M.) and Biological Andrology ( J.-C.S.),Assistance PubliqueHpitaux de Paris;and INSERM Unit 788 (P.L., M.S.) allat Bictre Hospital, Le Kremlin Bictre,France; Universit Paris VI, Departmentof Endocrinology and Reproductive Med-icine, PitiSalptrire Hospital, andCentre des Maladies EndocriniennesRares de la Croissance (C.C., A.B., F.K.,P.T.) and University Paris Descar tes (O.L.,

J.-C.S., M.M.) all in Paris; and AbdouHay Al Mahatta, Oujda, Morocco (H.B.).

Address repr int requests to Dr. Misrahi atINSERM Unit 854, Laboratory of Molecu-lar Genetics, Pharmacology, and Hor-monology, Assistance PubliqueHpi-taux de Paris, Bictre Hospital, 94275 LeKremlin Bictre, France, or at [email protected].

Drs. Achard, Courtillot, and Lahuna con-tributed equally to this article, as did Drs.Touraine and Misrahi.

N Engl J Med 2009;361:1856-63.Copyright 2009 Massachusetts Medical Society.

Summary

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 amutation 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. Hehad complete spermatogenesis with a normal sperm count. The mutant luteinizinghormone had a low level of partial activity in vitro. We concluded that the residualluteinizing hormone activity, resulting in the expression of steroidogenic enzymesin few mature Leydig cells producing small amounts of intratesticular testosterone(20.2 ng per gram), was sufficient for complete and quantitatively normal spermato-genesis.

Mutations 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 ofLeydig 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 onsetof normal spermatogenesis, which is thought to be critically dependent on a highlevel of intratesticular testosterone.6-9

We report a case of familial hypogonadotropic hypogonadism involving a partialloss of luteinizing hormone function. The proband had an undetectable circulating

luteinizing hormone level and a low serum testosterone level. He had a markedlysmall population of mature Leydig cells expressing the steroidogenic enzymesnecessary for androgen synthesis and producing low levels of intratesticular testos-terone. He had a mutant form of luteinizing hormone beta that showed low-levelresidual 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 triggerand maintain complete and quantitatively normal spermatogenesis.

The New England Journal of Medicine

Downloaded from www.nejm.org on October 4, 2010. For personal use only. No other uses without permission.

Copyright 2009 Massachusetts Medical Society. All rights reserved.


2/8

brief report

n engl j med 361;19 nejm.org november 5, 2009 1857

Case Report

This study was approved by the review boards ofthe institutions involved, and written informedconsent was obtained from the patient and hisfamily members. The 43-year-old proband (Sub-ject IV-3, with a 46,XY karyotype) (Fig. 1) had

been born in Morocco to consanguineous parentsand was previously treated there for hypogonad-ism at 28 years of age. Treatment with intramus-cular testosterone resulted in masculinization andpenile growth. The treatment was irregular be-cause of poor adherence; it had been discontin-ued for 3 months before the patient was referredto our department. Examination showed that thepatient was virilized (185 cm in height, 75 kg inweight) with normal masculine features: pubichair of Tanner stage 5, axillary hair of Tannerstage 3, penile length of 12 cm, normal testicular

volume, and absence of gynecomastia. Initial andsubsequent laboratory tests (Table 1) showed un-detectable luteinizing hormone levels (i.e.,


3/8


4/8

brief report


cells had an absence of anti-Mllerian hormone(Fig. 2I). The expression patterns of the androgenreceptor (Fig. 2J and 2K) and of both markers ofgerm-cell-maturation histone H1 (Fig. 2L and 2M)and proacrosin (Fig. 2N and 2O) were similar insamples from the patients testis and the controltestis.

Testosterone measurements were obtainedwith the use of gas chromatographymass spec-trometry.13 The level of intratesticular testoster-one in the patients biopsy sample (20.2 ng pergram of tissue) was approximately one eighteenththat of a specimen from an age-matched control(356.6 ng per gram of tissue); the control valuematches values in previous reports.14 Similarly,the level of intratesticular 5-dihydrotestosteronewas much less in the patients sample (0.59 ngper gram of tissue) than in the control sample(12.5 ng per gram of tissue) (Table S1 in the

Supplementary Appendix, available with the fulltext of this article at NEJM.org). The serum testos-terone level was 0.4 ng per milliliter (1.4 nmolper liter), confirming the immunoassay results.

The probands siblings (Fig. 1A) underwentnormal, spontaneous puberty. One sister, SubjectIV-5 (born in 1961), had menarche at 14 years ofage and subsequently had oligomenorrhea andsecondary amenorrhea. Repeated ultrasonogra-phy in the sister, for evaluation of infertility, at30 years of age revealed bilateral ovarian macro-cysts. Recent assays showed undetectable luteiniz-ing hormone and low estradiol and high follicle-stimulating hormone levels (Table 1). A brother,Subject IV-2 (born in 1967), was the father oftwo children, and another sister, Subject IV-4(born in 1975), had normal hormonal levels(Table 1). Yet another sister, Subject IV-1 (born in1957), was the mother of two children; she andthe other brother, Subject IV-6 (born in 1977),were unavailable for study.

Methods

DNA Sequencing

We sequenced the three exons ofLHB,as describedin the Supplementary Appendix.

Immunohistochemical Analysis

Immunohistochemical evaluation was performedwith the use of a commercial kit (LSAB+ systemwith 3-amino-9-ethylcarbazole, Dako), as describedpreviously.15

Functional Analysis of Mutant Luteinizing

Hormone Beta

Coding sequences of human LHA (encoding thealpha subunit of luteinizing hormone) and LHBwere inserted into the pSG5 vector (Stratagene).We then introduced the deletion carried by theproband into LHB to create a mutant luteinizing

hormone beta construct; we also introduced theV5 epitope into the C-terminal of the luteinizinghormone alpha subunit to make the construct-V5. Western blot analyses were performed withthe use of an antihuman chorionic gonadotro-pin antibody (ab14301, Abcam), which cross-reacts with the beta subunit of human luteinizinghormone; the samples blotted were cell lysatesand concentrated medium of human embryonickidney (HEK) 293T cells transfected with the useof a transfection reagent (SuperFect, Qiagen). Forcoimmunoprecipitation analysis, cell lysates from

transfected COS-7 cells (derived from fibroblastsfrom kidneys of African green monkeys) were in-cubated with anti-V5 antibody (Invitrogen), andimmunoprecipitates were analyzed by means ofWestern blotting. The activity of recombinant se-creted mutant luteinizing hormone relative to thatof wild-type luteinizing hormone was deter-mined by assessing cyclic AMP accumulation inHEK 293T cells transiently expressing the humanluteinizing hormone receptor. The level of wild-type luteinizing hormone in the culture mediumwas determined by means of immunofluoromet-ric assay (LHsp enzyme-linked immunosorbentassay, Biosource). Details are given in the Supple-mentary Appendix.

Results

Sequence analysis ofLHB from the proband (Fig.S1A in the Supplementary Appendix) revealed ahomozygous nine-base deletion in exon 2, pre-dicted to result in a deletion of amino acids 10 to12 (histidineprolineisoleucine) in the mutant

luteinizing hormone beta (Fig. 1B). Proline andthe 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 andIV-4) were heterozygous for the deletion (Fig. S1Bin the Supplementary Appendix).

Both the mutant and wild-type luteinizing hor-mone beta (both approximately 15 kD in size)16





5/8

Th e n e w e n g l a n d j o u r n a l o fmedicine


l

A B C

*

E F GD

I J KH

M N OL

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(arrowhead); there is also an isolated, mature Leydig cell in the interstitium (asterisk). (In Panels A, B, and C, stain-

ing was performed with hematoxylin and eosin.) In Panel D, only a few interstitial androgen-producing cells positivefor cytochrome P-450 (CYP) 17-hydroxylase are visible (arrows), whereas such cells are abundant in a sample from

an age-matched control (Panel E). We detected 3-hydroxysteroid dehydrogenase expression in both the few inter-stitial, mature Leydig cells also expressing CYP 17-hydroxylase (Panel F) and in the fibroblast-like precursors, adja-

cent to the tubular basement membrane and lacking CYP 17-hydroxylase (Panel G, arrows). Panel H shows Sertolicells strongly expressing anti-Mllerian hormone, whereas no such expression is observed in the control sample

(Panel I). (In Panels D through I, staining was performed with hematoxylin.) The expression pattern of the androgenreceptor 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 H1, a marker of germ-cell maturation, was observed in maturing germ

cells obtained from the patient (Panel L) and the control (Panel M). Proacrosin was detected in spermatids andspermatozoids 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 lowermagnification; Panels B, G, N, and O, twice the magnification; and Panels C and F, four times the magnification.





6/8

brief report


were synthesized in HEK 293T cells (Fig. 3A).However, we observed less mutant protein thanwild-type protein in cell lysates (mean [SD] ratioof 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 messengerRNA. In concentrated culture medium, the levelof wild-type protein (expressed as the mean [SD]of three independent experiments) was 67.98.6times that of the mutant protein (Fig. 3A). Afternormalization of this ratio on the basis of theintracellular expression ratio, we calculated thatthe 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 thealpha subunit in HEK 293T cells.

Coimmunoprecipitation studies with COS-7

cells cotransfected with expression vectors con-taining the -V5 construct and the wild-type ormutant luteinizing hormone beta subunit wereperformed to determine whether the defect insecretion of mutant luteinizing hormone beta re-sulted from defective subunit heterodimerization.Immunoprecipitates from cell lysates incubatedwith anti-V5 antibody, as previously described,2contained both subunits, indicating the occur-rence of heterodimerization of the alpha and beta

3000

CyclicAMP(

nmol/liter)

2000

2500

1500

150

50

100

0

40g

/liter

60g

/liter

120

g/lite

r

C

Stimulant Dose of LH

BA

l

Wild-type LHMutant LHMock-transfected

1 2 3 4

16

5 6

50

LH beta

Cell Lysate Culture Medium Anti-V5 AntibodyControl

22

16

50

1 2 3 4 5

kDkD

LH beta

-actin-actin

-V5

Immunoprecipitation

Cell Lysate

Mutan

tLH

beta

Wild-typ

eLH

beta

MutantL

Hbe

ta

Mock-t

ransfe

cted

Wild-ty

peLH

beta

MutantL

Hbe

ta

Mock-t

ransfe

cted

Wild-ty

peLH

beta

Mutan

tLH

beta

Wild-typ

eLH

beta

Wild-typ

eLH

beta

Figure 3. Production and Heterodimerization in Mutant and Wild-Type Luteinizing Hormone (LH) Beta and Bioactivity of Mutant

and Wild-Type LH.

Panel A shows low intracellular and secreted levels of mutant LH beta subunit in transfected cells. Western blot analysis was performedon cell lysate (lanes 1 through 3) and culture medium (lanes 4 to 6) of human embryonic kidney (HEK) 293T cells: cells producing theLH alpha subunit and the wild-type LH beta subunit (lanes 1 and 4), cells producing the LH alpha subunit and the mutant LH beta sub-

unit (lanes 2 and 5), and mock-transfected cells (lanes 3 and 6). An anti-actin antibody was used as a loading control (lanes 1 through 3).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

beta (lane 5), were loaded. Wild-type LH beta and mutant LH beta were immunodetected with an antihuman chorionic gonadotropin antibody (Abcam). Panel B shows low levels of dimerization of the alpha subunit and mutant beta subunit of LH. Coimmunoprecipita-

tion 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 with the use of anti-V5 antibody (lanes 4 and 5) or with a nonimmune immunoglobulin as a control(lane 3). This was followed by immunodetection of wild-type LH beta and mutant LH beta, with the use of a polyclonal antihuman cho-

rionic gonadotropin antibody (Abcam), or of the -V5 construct, with an anti-V5 antibody. An anti-actin antibody was used as a loadingcontrol (lanes 1 and 2). Panel C shows markedly lower levels of mutant LH bioactivity in HEK 293 cells expressing the human LH recep-

tor. The secreted levels of wild-type LH were quantified by means of immunofluorometric assay and used to generate a doseresponsecurve (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 receptorwere 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 experi-ments using each of three doses of mutant or wild-type LH to stimulate cyclic AMP production. I bars indicate standard deviations.





7/8

Th e n e w e n g l a n d j o u r n a l o fmedicine


subunits (Fig. 3B). However, immunoprecipitatescontained less mutant luteinizing hormone betathan the wild type (ratios in two independentexperiments, 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 thesecreted 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 was0.730.32% of that of the wild-type hormone.

Discussion

The patient described here had an absence ofspontaneous virilization, very low serum testos-terone levels, and minimal luteinizing hormone

activity but complete, quantitatively normalspermatogenesis.

In humans, there are three waves of Leydig-cell growth: the f irst, during the antenatal period,when growth is dependent on human chorionicgonadotropin; and the second and third, duringthe perinatal period and puberty, respectively,when growth is strictly under the control ofluteinizing hormone.9-11 Male infants displaytransient postnatal activation of the gonadotro-pin-releasing hormone pulse generator, inducinga surge in follicle-stimulating hormone, luteiniz-ing hormone, and testosterone that is correlatedwith normal adult spermatogenesis and fertility.17Partial stimulation of Leydig-cell proliferation,maturation, and function by luteinizing hormonemay have occurred in the testes of our patient,both perinatally and during puberty, to inducespermatogenesis. The mutant luteinizing hormonein our patient is known to retain partial activity,for two reasons. First, his testicular biopsy speci-men contained mature Leydig cells expressingthe steroidogenic enzymes CYP cholesterol-side-

chain 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, theintratesticular testosterone level in the patient(20.2 ng per gram) greatly exceeded (by a factorof 40) the serum testosterone level, a discrepancythat is consistent with testosterone productionwithin the testes.

The low production of intratesticular testos-

terone and consequent weak testosterone secre-tion into the serum (Table 1, and Table S1 in theSupplementary Appendix) were insufficient to in-duce virilization but were nevertheless capable,in cooperation with a functional follicle-stimulat-ing hormonegonadal axis, of exerting a localparacrine effect on contiguous Sertoli cells and

inducing the development and maturation ofseminiferous tubules that are required for com-plete spermatogenesis.18 Our findings support therecent proposal that congenital hypogonadotro-pic hypogonadism be managed both neonatallyand at puberty through the administration ofexogenous gonadotropins mimicking the physi-ologic gonadotropin surges.17,19 As has been de-scribed in men with other LHB mutations, ourpatient had inappropriately normal levels of in-hibin B, given his high plasma level of follicle-stimulating hormone.2,3 This may have been due

to the insufficient development of the follicle-stimulating hormoneinhibin feedback loop.20

The homozygous LHB deletion of the patientand two of his heterozygous, unaffected fam-ily members was found to occur near a disul-fide bridge forming the cystine-knot foldingmotif. This region may tolerate conformationalchanges.16

In male mice deficient in the luteinizing hor-mone receptor, spermatogenesis proceeds untilthe elongated spermatid stage, and the mice areinfertile.21 However, major differences in the hor-monal regulation of spermatogenesis are nowknown to exist between primates and rodents.Thus, to obtain complementary information onthe maintenance of spermatogenesis in normalmen undergoing gonadotropin suppression, stud-ies have been performed in men with the use ofhormonal contraception protocols. On adminis-tration of testosterone, both luteinizing hormoneand follicle-stimulating hormone are decreasedto minimal levels, suppressing intratesticular tes-tosterone production in these men.22,23 Intrates-

ticular testosterone levels similar to the serumlevels (3.81.3 ng per milliliter [13.24.5 nmolper liter])24 cannot maintain quantitatively normalsperm production. However, low intratesticulartestosterone 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 spermmaturation.22 Currently, the minimal intratesticu-lar testosterone level required to trigger and main-





8/8

brief report


tain normal sperm production in men is notknown.6,22

This study goes against current wisdom in thatit shows that complete and quantitatively normalspermatogenesis may be triggered and main-tained by low levels of luteinizing hormoneactivity postnatally and at puberty. A better un-

derstanding of the intratesticular hormonal micro-environment required throughout life (and espe-cially during the perinatal and peripubertalperiods) for adult spermatogenesis could resultin better strategies to treat certain patients withinfertility and to develop hormonal contraceptionmethods for men.22,23

Supported by grants from INSERM, the Socit FranaisedEndocrinologie, Assistance PubliqueHpitaux de Paris, andthe French Ministry of Health (DHOS).

No potential conflict of interest relevant to this article wasreported.

We thank G. Schaison, P. Roger, N. Lahlou, and E. Pussard forhelpful 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. Mailletfor help with sequencing analyses and polymerase-chain-reac-tionrestriction-fragmentlength polymorphism experiments;C. Coussieu, S. Brailly, and N. Lahlou for performing hormonalassays and S. Bart for performing the testicular biopsy; and A.Pianos, B. Eychenne, and A. Cambourg for technical assistancewith steroid determinations by means of gas chromatographymass spectrometry.

References

Weiss J, Axelrod L, Whitcomb RW,1.Harris PE, Crowley WF, Jameson JL. Hypo-gonadism caused by a single amino acid

substitution in the beta subunit of lu-teinizing hormone. N Engl J Med 1992;326:179-83.

Valdes-Socin H, Salvi R, Daly AF, et al.2.Hypogonadism in a patient with a muta-tion in the luteinizing hormone beta-sub-unit gene. N Engl J Med 2004;351:2619-25.

Lofrano-Porto A, Barra GB, Giaco-3.mini LA, et al. Luteinizing hormone betamutation and hypogonadism in men andwomen. N Engl J Med 2007;357:897-904.

Daly AF, Salvi R, Menage J-J, et al.4.Identification of a family harboring anovel LHb subunit mutation associatedwith hypogonadism. Presented at the

annual meeting of the Endocrine Society,Boston, June 2427, 2006. abst ract.

Valdes-Socin H, Salvi R, Thiry A, et al.5.Testicular effects of isolated luteinizinghormone deficiency and reversal by long-term human chorionic gonadotropin treat-ment. J Clin Endocrinol Metab 2009;94:3-4.

McLachlan RI, ODonnell L, Meachem6.SJ, et al. Identification of specific sites ofhormonal regulation in spermatogenesisin rats, monkeys, and man. Recent ProgHorm Res 2002;57:149-79.

Dohle GR, Smit M, Weber RF. Andro-7.gens and male fertilit y. World J Urol 2003;21:341-5.

Lombardo F, Sgr P, Salacone P, et a l.8.Androgens and fertility. J Endocrinol In-vest 2005;28:Suppl:51-5.

Wu X, Wan S, Lee MM. Key factors in9.the regulation of fetal and postnatal Ley-dig cell development. J Cell Physiol 2007;

213:429-33.Guzick DS, Overstreet JW, Factor-10.Litvak P, et al. Sperm morphology, motil-ity, and concentration in fertile and infer-tile men. N Engl J Med 2001;345:1388-93.

Saez JM. Leydig cells: endocrine, para-11.crine, and autocrine regulation. EndocrRev 1994;15:574-626.

Lewis K, Lee PA. Endocrinology of12.male puberty. Curr Opin Endocrinol Dia-betes Obes 2009;16:5-9.

Meffre D, Pianos A, Liere P, et al. Ste-13.roid profiling in brain and plasma of maleand pseudopregnant female rats after trau-matic brain injury: analysis by gas chro-matography/mass spectrometry. Endocri-

nology 2007;148:2505-17.McLachlan RI, ODonnell L, Meachem14.

SJ, et al. Hormonal regulation of spermato-genesis in primates and man: insights fordevelopment of the male hormonal con-traceptive. J Androl 2002;23:149-62.

Meduri G, Bachelot A, Cocca MP, et al.15.Molecular pathology of the FSH receptor:new insights into FSH physiology. MolCell Endocrinol 2008;282:130-42.

Huhtaniemi IT, Themmen AP. Muta-16.tions in human gonadotropin and gonado-tropin-receptor genes. Endocrine 2005;26:207-17.

Grumbach MM. A window of oppor-17.tunity: the diagnosis of gonadotropin de-

ficiency in the male infant. J Clin Endo-crinol Metab 2005;90:3122-7.

Plant TM, Marshall GR. The function-18.

al signif icance of FSH in spermatogenesisand the control of its secretion in maleprimates. Endocr Rev 2001;22:764-86.

Bougnres P, Franois M, Pantalone19.L, et al. Effects of an early postnatal treat-ment of hypogonadotropic hypogonadismwith a continuous subcutaneous infusionof recombinant follicle-stimulating hor-mone and luteinizing hormone. J ClinEndocrinol Metab 2008;93:2202-5.

Andersson AM, Juul A, Petersen JH,20.Mller J, Groome NP, Skakkebaek NE. Se-rum inhibin B in healthy pubertal andadolescent boys: relation to age, stage ofpuberty, and foll icle-stimulating hormone,luteinizing hormone, testosterone, andestradiol levels. J Clin Endocrinol Metab1997;82:3976-81.

Zhang FP, Pakarainen T, Poutanen M,21.

Toppari J, Huhtaniemi I. The low gonado-tropin-independent constitutive produc-tion of testicular testosterone is sufficientto maintain spermatogenesis. Proc NatlAcad Sci U S A 2003;100:13692-7.

Page ST, Amory JK, Bremner WJ. Ad-22.vances in male contraception. Endocr Rev2008;29:465-93.

Kamischke A, Nieschlag E. Progress23.towards hormonal male contraception.Trends Pharmacol Sci 2004;25:49-57.

Coviello AD, Bremner WJ, Matsumoto24.AM, et al. Intratesticular testosterone con-centrations comparable with serum levelsare not sufficient to maintain normalsperm production in men receiving a hor-

monal contraceptive regimen. J Androl2004;25:931-8.Copyright 2009 Massachusetts Medical Society.

collectionsofarticlesonthejournalswebsite

TheJournals Web site (NEJM.org) sorts published articles intomore than 50 distinct clinical collections, which can be used as convenient

entry points to clinical content. In each collection, articles are cited in reversechronologic order, with the most recent first.



Copyright 2009 Massachusetts Medical Society All rights reserved

normal spermatozoa

Documents