212 THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 3, NO.5

Testicular histology and gonadotropin levels in infertilemen with non-obstructive oligo-/azoospermiaARUN KUMAR, B. JAYAKUMAR, M. L. KHURANA, VED PRAKASH,M. VIJA YARAGHAVAN, N. K. SHUKLA, A. C. AMINI,M. G. KARMARKAR, M. M. S. AHUJA

ABSTRACTThe common histological abnormalities on testicularhistology in 79 infertile virile men were hypospermato-genesis (35%), severe testicular atrophy (29%) andmaturation arrest (19%). Sperm counts showed no correla-tion with testicular size, gonadotropin levels and histologicalclassification. There was elevation of mean folliclestimulating hormone levels in males with maturationarrest (5.8±3.8 lUlL) and Sertoli-cell only syndrome(21.7±3.4 lUlL). Patients with severe testicular atrophyhad elevated luteinizing hormone (15.1±11.8 lUlL) aswell as follicle stimulating hormone (49.8±11.4 lUlL)levels. Patients with hypospermatogenesis and focalatrophy had normal luteinizing hormone and folliclestimulating hormone levels and all of them had normaltestosterone and prolactin levels. Treatment withgonadotropins or clomiphene citrate was ineffective.

INTRODUCTIONMarried couples are considered infertile if they fail toachieve conception during at least one year of unprotectedintercourse. Although the prevalence of infertility isdifficult to assess, it has been estimated that about 15% ofmarried couples are infertile. The male factor alone isresponsible for a third of infertile couples and in anotherthird the male factor plays a contributory role. 1-3 Malesmay be absolutely infertile owing to an absence of spermsin the semen (azoospermia) or partially infertile owing toa paucity of sperm number (oligospermia). The hormonalcontrol of spermatogenesis is mediated by the directaction of hormones on somatic cells but not on germcells." Interactions of germ cells with Sertoli, Leydig andperi tubular cells play an important role in local regulationof testicular function+" Follicle stimulating hormone(FSH) and androgens, by complex modulation of gonadal

All India Institute of Medical Sciences, New Delhi 110029, IndiaARUN KUMAR, B. JA YAKUMAR, M. L. KHURANA,

VED PRAKASH, A. C. AMINI, M. G. KARMARKAR,M. M. S. AHUJA Department of Endocrinology and Metabolism

M. VIJA YARAGHA VAN Department of PathologyN. K. SHUKLA Department of Surgery

Correspondence to M. G. KARMARKAR

© The National Medical Journal of India 1990

somatic cells, create an optimal micro-environment forspermatogenesis within the seminiferous tubules." Seminalanalysis forms an important screening test for assessmentof the fertility potential of males. In this study we correlatedthe clinical profile, results of semen analysis, gonadotropinlevels and testicular histology to assess their relativeimportance in the evaluation of infertile men.

MATERIALS AND METHODSSeventy-nine consecutive infertile virile males withnon-obstructive oligo- or azoospermia referred to theEndocrine Clinic of the All India Institute of MedicalSciences between September 1987 and September 1989were studied. All patients had a careful history taken andhad a thorough physical examination. Particularattention was paid to a history of specific childhood ill-nesses such as cryptorchidism, postpubertal mumps,orchitis, testicular trauma and torsion, exposure tooccupational and environmental toxins and exposure toexcessive heat or radiation. We noted whether drugs suchas anabolic steroids, cimetidine and spironolactone hadbeen consumed and whether narcotics and alcohol hadbeen taken. A history of chronic systemic and endocrinedisease including loss of libido and impotence was alsorecorded. Special attention during examination was paidto skeletal proportion, pattern of hair distribution,muscular mass and development, characteristics of thegenitalia and whether a varicocele was present. Allsubjects had the following investigations: semen analysis,luteinizing hormone (LH), FSH, testosterone, prolactin,blood sugar, VDRL levels, X-rays of the chest and skulland testicular biopsy. The testicular size was measuredusing a Prader's orchidometer.

The histological changesv':" in the testes were classifiedto be as follows.

Normal, when examination showed seminiferoustubules containing germ cells and Sertoli cells lined bybasement membrane with normal spermatogenesis withsperms in different stages of maturation (Fig. 1).

Hypospermatogenesis, when there was an overallreduction in the number of germ cells in all stages ofspermatogenesis. The changes might vary from tubule to

KUMAR et at. : TESTICULAR HISTOLOGY

FIG 1. Normal testicular biopsy showing seminiferous tubuleswith normal basement membranes and normal maturationof spermatocytes (H&E, x 100)

FIG 3. Testicular atrophy. The seminiferous tubules are totallyhyalinized. No germ cells or Sertoli cells are seen(H&E, x 100)

tubule and be associated with focal peri tubular and inter-stitial fibrosis. The Leydig cells were unaffected (Fig. 2).

Maturation arrest, indicated by cessation of spermato-genesis at some stage of maturation. Arrest at differentstages of maturation might be observed in different partsof the testes. The Leydig cells were normal.

Focal atrophy, when there were localized areas in theseminiferous tubules with partial or complete obliterationof the lumen, thickening of basement membrane and anabsence of germ and Sertoli cells.

Severe testicular atrophy, when there was a marked lossof germ cells with fibrous obliteration of the tubules,hyalinization of the basement membrane and a character-istic nodular 'adenornatous' proliferation of the Leydigcells (Fig. 3).

Sertoli-cell only syndrome was diagnosed when no germcells were seen while the Leydig and Sertoli cells werenormal. The basement membrane is normal in the con-

213

FIG 2. Hypospermatogenesis. Normal germ cell maturation isseen in occasional tubules (H&E, x 100)

FIG 4. Sertoli-cell only syndrome. Seminiferous tubules arelined by Sertoli cells. No germ cells are present. Thebasement membrane is normal (H&E, x 100)

genital type (Del Castillo syndrome) and thickened in theacquired type of the syndrome (Fig. 4).

The subjects with infertility were divided into twogroups. One group (30 subjects) was treated for at leastsix months in 30-day cycles and given clomiphene citrate50 mg daily for 25 days with a drug-free period of fivedays. The second group (21 subjects) was given humanchorionic gonadotropin (hCG) 2000 units twice a week fora minimum period of six months. Five subjects with theSertoli-cell only syndrome and 23 subjects with severetesticular atrophy were not treated.

RESULTSSeventy out of the 79 males with infertility had primarysterility and nine secondary ste:ility. The mean age of thesubjects was 31±4.7 years. Subjects with primary sterilitycame after 6.1 ±4.4 years of childless marriage while sub-jects with secondary sterility came 8.2±3.5 years after the

214

TABLE I. Historical details and histological correlation

THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 3, NO.5

Normal Maturation Hypospermato-arrest genesis

Severe testicularatrophy

History n Sertoli-cellonly syndrome

Unilateral small testes' 6 2 2Hydrocele (optd.) 5 2 1Mumps orchitis 3 1 1Trauma 2Epididymo-orchitis 2Varicocele (optd.) 1Cryptorchidism RI. (optd.) 1Leprosy 1

• Only bigger testis biopsied optd operated

TABLE II. Correlation of testicular volume and oligo- or azoospermia with histological classification

Total(n=79)

Normal-sized testes(testicular volume> 15ml)

Small-sized testes(testicular volume < 10ml)

HistologyOligospermia

(n=20)Azoospermia

(n=34)

3 21

Normal 6Focal atrophy 1Maturation arrest 15 3Focal atrophy and maturation 1

arrestHypospermatogenesis 28 13Sertoli-cell only syndrome 5Severe testicular atrophy 23 3

Oligospermia(n=9)

Azoospermia(n=16)

111

1036

3 22

113

TABLE III. Correlation of sperm counts with histologicalclassification

Histology Total Sperm count (106/ml)(n=79) <1 Nil 1-4.9 5-20

Normal 6 3Focal atrophy 1Maturation arrest 15 12Focal atrophy and matura- 1 1tionarrest

Hypospermatogenesis 28 12 5 2Sertoli-cell only syndrome 5 5Severe testicular atrophy 23 17 3 3

2

9

TABLE IV. Histological classification and gonadotropin levels

Histology Total LH FSH(n=79) (2.5-9.8 lUlL) (1.2-5.0 lUlL)

Normal 6 6.7±2.7 4.4±2.6Focal atrophy 1 9.6 2.7Maturation arrest 15 6.O±4.1 5.8±3.9Focal atrophy with maturation 1 8.6 2.2arrest

Hyposperrnatogenesis 28 9.0±6.3 4.9±5.3Sertoli-cell only syndrome 5 8.8±4.1 21.7±13.4Severe testicular atrophy 23 15.1±11.8 49.8±11.4

last childbirth. Subjects with secondary sterility reportedfor evaluation because they had either only one child orhad had two daughters. Seven subjects with primary infer-tility appeared to have a eunuchoidal body habitus,however, karyotype revealed an XY pattern in all. Theimportant historical and clinical details are provided inTable I.

Twenty-five subjects had small testes (volume <10 ml)and in nine of these subjects the testicular atrophy wasmore marked on one side. Azoospermia was present in50 subjects and 11 subjects had counts either less than1 million/ml or had occasional sperms seen in the semen(Tables II and III). The semen was positive for fructose inall the patients.

Histological hypospermatogenesis (35%), severetesticular atrophy (29%) and maturation arrest (19%)were the predominant abnormalities (Table II). Signifi-cant elevation of FSH (21.7±13.4 lUlL) with normalLH (8.8±4.1 lUlL) levels were observed in subjects withthe Sertoli-cell only syndrome. However, in subjects withsevere testicular atrophy significant elevation of LH(15.1±11.8 lUlL) as well as FSH (49.8±11.4 lUlL) wereobserved. Hypospermatogenesis and focal atrophy wereassociated with normal LH and FSH levels, except for twosubjects with severe hypospermatogenesis who had ele-vated FSH (8.6 and 10.2 lUlL) levels. Mean FSH levels(5.8±3.86 lUlL) were elevated in subjects with matura-tion arrest (Table IV). Prolactin (159±64.3 mIUIL) and

KUMAR et III. : TESTICULU HISTOLOGY

LHIUIl

50

.' .

40 ....

30

20

..'10 1---~"7'~----------~------------~----NormoI ,- :.:

rwtgI :.: ••

O~--~------~------.--------r------~o 5 10 15

Spann counts (1CJI'/mI)

FIG5. Correlation of LH levels with sperm counts

testosterone (26±8.4 nmol/L) levels were normal in allsubjects. No correlation was found between sperm countand gonadotropin levels (Figs. 5 and 6).

One subject with the Sertoli-cell only syndrome hadvery little seminal discharge. Sonography and retrogradegenitourethrography revealed a strictured urethra withpost-void residual urine. There was no correlation betweenoligospermia or azoospermia and any histological classifi-cation except the Sertoli-cell only syndrome. Azoospermiawas more often seen in severe testicular atrophy withsmall-sized testes and maturation arrest with normal-sizedtestes (Table II). Lymphocytic infiltration of testiculartissue was seen in one subject each with hypospermato-genesis, maturation arrest and severe testicular atrophy.This probably indicates previous viral or autoimmuneorchitis.

Clomiphene citrate or hCG failed to stimulate sper-matogenesis in .six months in the azoospermic males andthere was no significant improvement in sperm counts(pre-treatment level 8.2±7.1 x l06/ml, post-treatmentleveI8.6±7.2 x l06/ml) in oligospermic males. However,pregnancy was confirmed in the wife of one subject witholigospermia secondary to hypospermatogenesis afterfour months of stimulation with hCG.

DISCUSSIONA primary endocrine defect of the gonads is rare in infertilemales with a sperm count of more than 5 million/mi. \0But secondary changes in the pituitary or hypothalamusaffecting gonadotropins may be seen in subjects witholigospermia and azoospermia. There is a correlationbetween FSH levels and sperm counts when the count isless than 5 million/ml'! and an isolated increase in thelevel of FSH together with a low sperm count indicatesgerm cell destruction or aplasia."

Maturation arrest, another common disorder, may beassociated with normal or elevated FSH levels. 12 Matura-tion arrest has been presumed to be the result of severe

215

FSHIUIl

eo

· ..

50· .

40

30

20 .':.· ..10

NormoI I----~,,~.~.~--------~~------------------~ol=~·~:·======~====·=·~:·======~=====;20 15 205 10o

Spann 00<.f1Is (1 CJI'/mI)

FIG6. Correlation of FSH levels with sperm counts

hypospermatogenesis with irregular distribution.PMaturation arrest at the spermatid stage is seen in subjectswith cryptorchidism and chronic inflammation of thereproductive organs. 14

A varicocele, commonly seen in the left scrotal sac, mayalso be associated with oligospermia. Unilateral varicoce\esare associated with morphological changes in the contra-lateral testes as well. 15There is no correlation between thesize of a varicocele and sperm count or testicular histology"and return of fertility after repair of a varicocele isunpredictable. 16.17

Our patients were virile men with normal plasmatestosterone and prolactin levels. Elevated gonadotropinlevels were observed in patients with severe testicularatrophy. An isolated elevation of FSH was observed inpatients with the Sertoli-cell only syndrome and severehypospermatogenesis. In others we found no correlationbetween sperm counts and levels of gonadotropin. Therewas also no correlation between sperm counts and histo-logical classification. Histological appearances rangingfrom normal to near total loss of germinal epitheliumwere observed in both oligospermic and azoospermicindividuals and also in subjects with normal and small-sized testes. This is in accordance with previous reports. 7.8.13All patients with the Sertoli-cell only syndrome hadazoospermia. Histologically 4 of the 5 patients withSertoli-cell only syndrome had a thickened basementmembrane, indicating they had the acquired variety ofthis disorder. Three of them had had epididymo-orchitis,mumps and chickenpox in the past. The fourth, it wrestler,had been using a langot (tight underwear) for many years.

Three patients with azoospermia had normal testicularhistology and similar cases have been reported earlier.7.8·18Since fructose positivity of the semen excludes a blockbeyond the seminal vesicles, these subjects may have hadobstruction in or around their epididymis or they mayhave had severe hypospermatogenesis.

216

Hypospermatogenesis has been the commonest histo-logical feature in most reported studies of infertile menwith or without varicocele+?" although Agarwal et al.xfound local atrophy to be more common. 35% of ourpatients had hypospermatogenesis, 29% had severetesticular atrophy and 19% had maturation arrest.

Since elevation of LH and or FSH indicates severetesticular atrophy or marked destruction of the germinalepithelium the likelihood of fertility is poor and testicularbiopsy is not indicated. Azoospermia with normal histo-logy or mild tubular damage with a considerable numberof germ cells suggests post-testicular obstruction, apotentially reversible condition." The presence of anadequate number of germ cells in the biopsy and exclusionof post-testicular obstruction identifies a group with afavourable prognosis for fertility. 20

Clomiphene citrate by increasing LH, FSH and testo-sterone may be effective in providing additional germinalepithelium.' hCG and human menopausal gonadotropin(hMG) stimulate testicular tissue directly. An increase insperm counts from 1million/ml to 16million/ml with hCGstimulation resulted in pregnancy in one of our couplesafter four years of married life. However, spontaneouspregnancies in oligospermic couples also occur. Males withidiopathic oligospermia may have a subtle abnormality ordeficiency in gonadotropin production, or a form of insen-sitivity of Leydig cells to normal levels of gonadotropins,requiring higher than normal circulating levels. IClomiphene induced elevation of gonadotropins may bebeneficial in these subjects. However, the overall resultsof treatment with gonadotropins or gonadotropinstimulating agents have been disappointing-l-P with a fewrandom reports showing a positive response. 22.23 It is notknown whether treatment for longer than six monthsmight be more beneficial. Severely atrophic testes withscanty germ cells are probably incapable of regenerationfrom the remaining spermatogonia. Those with theSertoli-cell only syndrome or with tubular hyalinizationhave lost germ cells and so also the ability to regenerate.These subjects are severely affected and treatment doesnot seem to be worthwhile. 18

We conclude that elevated FSH levels in oligo- orazoospermic males indicates a poor prognosis for futurefertility. Oligospermic males with normal FSH levels maybe stimulated using clomiphene citrate or hCG.

THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 3, NO.5

REFERENCESSteinberger E. Male infertility. In: Gold JJ. Josimovich JB (eds).Gynaecologic Endocrinology. New York:Plenum Medical Books.1987:551--90.

2 Hull MGR. Glazener CM. Kelly NJ. et al. Population study ofcauses. treatment and outcome of infertility. Br Med J 1985;291:1691--7.

3 Jones TM. Infertility. In: DeGroot LJ (ed). Endocrinology.Philadelphia:WB Saunders. 1989:2192-206.

4 Martinova Y. Kontcheva L. Tzvetkou D. Testicular ultrastructurein infertile man. Arch AndroI1989;22:101--22.

5 Sharpe RM. Local control of testicular function. Q J Exp Physiol1983;68:265-87.

6 Sharpe RM. Intratesticular factors controlling testicular function.Bioi Reprod 1984;30:29-49.

7 Leong AS. Mathews CD. The role of testicular biopsy in the investi-gation of male infertility. Pathology 1982; 14:205-10.

8 Agarwal S. Sikka M. Dev G. Banerjee A. Testicular size. morphologyand spermatogenesis in infertile males with varicocele. lnd J PatholMicrobioI1988;31:19-25.

9 Mostofi KF. Davis CJ Jr. Male reproductive system and prostate.In: Kissane JM (ed). Anderson's Pathology. St Louis:CV Mosby.1985:791-832.

\0 Dubin L. Amelar RD. Etiologic factors in 1294 consecutive cases ofmale infertility. Fertil Steril1971 ;22:469-74.

II Hargreave TB. Jequier AM. Can FSH estimation replace testicularbiopsy in the diagnosis of obstructive azoospermia. Br J Urol1978;50:415-18.

12 deKrester OM. Endocrinology of male infertility. Br Med Bull1979;35:187-92.

\3 Levin HS. Testicular biopsy in the study of male infertility: Itscurrent usefulness. histologic techniques. prospects for the future.Human PathoI1979;10:569-84.

14 Holstein AF. Ultrastructural observations on the differentiation ofspermatids in man. Andrologia 1976;8: I57-{i5.

IS Dubin L. Amelar RD. Varicocele. Urol c/in N Am 1978;5:563-72.16 Marks JL. McMahon R. Lipschultz LI. Predictive parameters of

successful varicocele repair. J UroI1986;136:609-12.17 Baker HWG. Burger HG. de Krester OM. et al. Testicular vein

ligation and fertility in men with varicoceles. 8r Med J 1985;291:1678-80.

18 Meinhard E. McRae CV. Chisholm GO. Testicular biopsy in evalua-tion of male infertility. 8r Med J 1973 ;3:577-81.

19 Sauten RJ. The testes. In: Felig P. Boxter JD. Broodus AE.Frohmen LA (eds). Endocrinology and Metabolism. New York:McGraw-Hili. 1987:821-905.

20 Cunnigham GR. Medical treatment of the subfertile male. UrolClin N Am 1978;5:537-48.

21 Foss GL. Tindall YR. Birkett JP. The treatment of subfertile menwith clomiphene citrate. J Reprod FertilI973;32: 167-70.

22 Steinberger E. Medical treatment of male infertility. Andrologia[Suppl] 1976;8:77-9.

23 Paulsen OF. Wacksman J. Hammond CB. et al. Hypofertilityandclomiphene citrate therapy. Fertil Steri/1975;26:982-7.