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9th Postgraduate Course for Training in Reproductive Medicine and Reproductive Biology

Etiology of male infertility and Oligo-, Astheno-, Teratospermia (OAT)

R.C. Martin-Du Pan
(Arch Androl 39:197,1997)



VI. IMPAIRMENT OF SPERM TRANSPORT: obstructive azoospermia




What is a normal sperm ?

According to the WHO guidelines, minimum sperm values are 2 ml (volume), 20 million/ml (concentration), 50% (motility) and 30% (normal morphology). However, there is no exact threshold under which sperm values can be considered abnormal. Some authors claim that fertility decreases only with a sperm concentration of 5 million/ml or less (Jouannet, Ann Biol Clin 45:335;1987). Using the 10th percentile Ombelet (Hum Rep 12:987,1997) has found a cut-off value of 14 million/ml for sperm concentration, 28% for progressive motility, 8 million for total motile sperm count and 5% for sperm morphology using the strict criteria of Kruger. Normal values for sperm morphology depend on the classification method (Chia, Hum Rep 13:3394,1998). There is a large fluctuation of sperm values depending on the duration of abstinence, the conditions of sperm collection and the season and possibly also the time of the day (Cagnacci, Hum Rep 14:106,1999)

(Hum Rep 14:1028,1999; J Andr 14:366,1993; F St 49:309,1988).

This subject will be discussed in more detail in the lecture `Semen analysis`.


1.Conditions of semen collection

  • incomplete sperm collection
  • use of condoms (spermicids),vaginal lubricants
  • duration of sexual abstinence: short - oligospermia (Hum Rep 14:1028 1999), increased motility (Hum Rep 8:1251,1993); long- asthenospermia

2. Environmental factors

  • occupational heat exposure (Hum Rep 13:2122, 1998)
  • sauna, hot baths,tight underwear (J Androl 20:18;1999)
  • feverish states
  • season: oligospermia in September (heat, photoperiodicity ?)
  • toxic products: lead, cadmium (Am J Epidemiol 135:1208,1992)
  • dibromochloropopane (DBPC), kepone
  • boric acid, vinyl chloride
  • aromatic solvents (F St 71:690,1999)
  • drugs: heroine, methadone: FSH and LH ( marijuana: T and sperm mobility)
  • alcohol: inhibition of T synthesis and sperm capacitation,
  • reduced sperm quality in heavy drinkers (Andrologia 3:43,1999)
  • cigarette smoking: in 11 studies the sperm density is 22% lower in smokers, the motility is lower (F St 46:545,1986); increased sperm disomy has been found (F St 70:715,19 98)

3. Iatrogenic factors

Surgery: hernioraphy in childhood (lesion of vasa), testicular torsion, orchidopexy

  • radiotherapy: B spermatogonia are sensitive to radiation. Permanent sterility for doses 600-800 rad (F St 45:443, 1986)
  • chemotherapy: alkylating agents (cyclophosphamide = antimitotic) 75 to 100 % azoospermia after MOPP. Less with ABVD

No protection by GNRH.

Drugs : with antiandrogenic action: spironolactone, cimetidine, ketoconazole, cyproterone acetate, tetracycline, phenytoin, carbamazepine (BMJ 284:844, 1982)

  • gonadotrophins: estrogens, androgens
  • sulfasalazyne, furodantoines, garamycine.
  • possible adverse effect: cotrimoxazol,antimalarials, amoebicides, tetracyclines (Corinne, Hum Rep 13:1878,1998) - impotence or ejaculation disorders: neuroleptics, thioridazine,
  • clomipramine, alpha and beta adrenergic blocking drugs,clonidine, methyldopa, fibrates, anticholinergics etc, SSRI (fluoxetine,citalopram)
  • drugs without adverse effect on sperm: ranitidine, 5 -aminosalicylique, amitryptiline, enzyme conversion inhibitors (enalapril), AINS (diclofenac), quinolones (ciprofloxacine)


1. Infection

  • Orchitis: mumps, echovirus, B arbovirus. Mumps: 75% of men with unilateral orchitis have a normal sperm within 1 to 2 years but only 30 % with a bilateral orchitis (Bartak, Int J Fertil 13:226,1968; Shulman, F St 57:1344,1992). Treatment with interferon 2B ?(J Urol.146:54,91)
  • Epididymitis: Chlamydia, Gonococci, Tuberculosis: obstruction of epididymis: azoospermia + antisperm antibodies (Hendry, Brit J Urol 146:54,1983)
  • Prostatitis: present in 25 to 30% of infertile compared to 10-15 % of fertile men. Controversial cause of asthenospermia and male infertility. Leukospermia and decreased seminal levels of zinc. Possible role of mycoplasma, chlamydia, capable of attaching to sperm. Induction of autoantibodies.Controversial effect of antibiotics (Keck, Hum Rep Update,4:891,1998).
  • HIV: present in 10 to 30% of the sperm of  seropositive men ( in 87% by PCR). The virus is present in semen as cell-free or as cell-associated virus (mononuclear cells) (Kiessling, F St 58:667, 1992). Insemination with processed semen of HIV-partners is still experimental and controversial (Semprini, Lancet 340,1317,1993; Edlin, Lancet 341:570,1993). The virus is still present in seminal cells of patients receiving antiretroviral therapy (NEJM 339:1803,1998).

2. Immunologic causes: antisperm antibodies (Mazumdar, F St 70:799,1998)

Antisperm antibodies have been detected in 5 to 10% of infertile men and in 2% of fertile men. Antibodies against spermatozoa can reduce fertility by decreasing the binding of sperms to the zona pellucida,, by interfering with capacitation or acrosome reaction or by immobilising sperms in cervical mucus. Antibodies directed against the sperm head are deleterious. The presence of antisperm antibodies is determined by the attachment of a labelled antihuman immunglobulin  specific for the class of human Ig to be essayed, to the sperm-associated antibody. The label can be an erythrocyte (MAR test), a polycramid bead (immunobead essay) an enzyme (ELISA), a fluorescent molecule or a radioisotope (Krapez, Hum Rep 13:3363,1998).

a) Male autoimmunisation

- Antisperm autoantibodies in semen:

They induce no specific abnormality in sperms but could decrease the mobility and can be detected by MAR test or immunobead essay (IgG or IgA). The cause is unknown: possible cross reactivity betweeen sperm internal antigens and certain microorganisms ? An increased incidence has been found in the case of associated prostatitis. Antisperm antibodies are also present after vaso-vasostomy, inversely related to sperm motility (Broderick, J Urol 142:752,1989). Antibodies (Ab) have a high affinity for sperm surface antigens and they cannot be removed by washing (even 18 times). Ab could be added by exposure to seminal plasma and ejaculation into buffer can be useful to decrease antibody-bound sperm by dilution (Alexander, F St 53:602,1990). IVF is possible except in case of antibodies directed against sperm head or acrosine (Zouari, F St 59:606,1993), ICSI is preferred in the latter situation (Mazumdar, F St 70:799,1998).

- Circulating antisperm autoantibodies

Unbound antibodies can be measured by the tray agglutination test (serial dilutions of serum are able to agglutinate donor spermatozoa) or by the indirect immunobead test (antisperm antibodies in the serum will bind to donor sperm free of antibody). Ab can be produced because of disruption of the blood testis barrier that isolate sperm antigens from the male's immunolgical system (testis biopsy, torsion). Leakage of sperm due to sperm degeneration in the epididymis could be responsible for the occurrence of circulating antibodies in unilateral or bilateral obstruction of the male genital tract (e.g. antibodies are present in 50 to 80 % of cases after vasectomy) (F St 46:753,1986). Genetic factors (HLA A28) could play a role. The role of the circulating ab in infertile couple and in the persistent infertility after vaso-vasostomy as well as the utility of prednisone treatment are controversial (Hum Reprod 13:3363,1998; F St 70:799,1998).

- Autoantibodies in the testis:

Autoantibodies directed against testicular basement membrane and against steroid cells (Leydig cells) have been described in rare cases of hypogonadic men with multiple endocrine autoimmune diseases (Murthy, JCEM 42:637,1986 and 52:1137,1981).

c) HLA sharing in couples

Recurrent spontaneous abortion and failure of IVF are more frequent in women who share HLA-B,DR and DQ antigens with their husbands (Jin, Am J Med Genet 56:1456,1995).

3. Varicocele

The incidence is 10-15% in the male population and 20 to 30% in infertile patients. Varicoceles are associated with impaired seminal and hormonal parameters.  It is a classical cause of secondary infertility. Adverse effects could result from increased scrotal temperature, reflux from the adrenal gland or adrenal metabolites (left internal spermatic vein enters the left renal vein). A review of 509 publications comprising 5471 patients shows that surgical ligation of the spermatic vein results in an average pregancy rate of 36% (Mordel, J Reprod Med 35:123,1990). However the benefit of surgery has not been proved in a randomized study controlling for female factors (29% pregnancy in the treatment group and 25% in the non- treatment group (n=125)(Nieschlag, Hum Rep 13:2147,1998).

Practically, varicocelectomy can be recommended in order to prevent a further detorioration of the sperm if the following criteria are fulfilled (Nieschlag, Clin Endocrinol 38:123,1993):    

  1. infertility for 1 year
  2. Valsalva-positive varicocele documented by doppler and US
  3. smaller testicular volume on the varicocele side
  4. subnormal seminal parameters
  5. FSH not elevated
  6. normal or treatable female reproductive functions
  7. no other causes of male infertility (prostatitis, autoimmunity etc)

High surgical ligation (by laparoscopy !) and angiographic embolization give similar results.

4. Testicular tumors:

Testicular tumors affect 2-3/100.000 men per year and are responsible for 1% of cancer deaths.  Men with cryptorchidism have a fivefold increased risk (3/4 are seminoma= tumors arising from the germinal epithelium). Tumor of adrenal cell rests dependent on ACTH have been described in 21-hydroxylase deficiency and can decrease after corticoid treatment (Cutfield, 1983). About 50% of men with germ cell tumors have initial low sperm count (J Urology 126:141, 1981). Carcinoma in situ (CIS) has been found in 0,4% to 1,1% of infertile male (Pryor, Brit J Urol,55:780,1983). CIS are more frequent in infertile patients (Giwerkman, Int J And 10:173,1987; Novero, F St 65:1051,1996).


1.Cryptorchism (Hutson End Rev 18:259,1997)

Cryptorchism exists in 0,7-0,8% of adult men and in 2-3% in newborns and is present in 6% of infertile patients. Maldescent occurs in more than 40 human congenital defects including cases of hypogonadism and lack of androgen synthesis or action. It is associated with HLA-A11 and A-25. The lack of descent after HCG occurs in 40% of HLA-A11 and 70% of HLA-DR5 (Martinetti, JCEM 74:39,1992). It is not clear whether the testis functions poorly because of the  maldescent (heat etc.) or it fails to descent because it is initially abnormal. Spermatogenesis is also abnormal in the descended testis. Deleterious changes in the ultrastructure of the cryptorchid testis are observed in the first year of life (J Urol 128:782,1982). Therefore it has been suggested to operate the cryptorchid testis in the first year in case of lack of reponse to GnRH or HCG (Canavese, Ped Surg Int 14:2,1998). However, operation may not ameliorate the fertility potential in cases of bilateral cryptorchism. In case of unilateral cryptorchism surgery in early puberty has been advised by some authors (Okuyama, J Urol 142:749,1989; Lenzi F St 67:943,1997).

In bilateral cryptorchism 42% of treated patients are azoospermic and 31% are oligospermic. In untreated cases 75% are azoospermic. In unilateral cases 14% of treated patients are azoospermic and 31% are oligospermic (the results are not different for treated cases) (Chilvers, J Ped Surg 21:691,1986).

2. Genetic causes (Johnson F St 70:397,1998)

- Karyotype abnormalities have been observed in 15 to 23% of azoospermic and in 5-6% of oligospermic patients (Rivas, J Genet Hum 35:291,1986;  Bourroullon, Hum Genet 71:336,1985).

- Klinefelter syndrome (XXY):

Frequency: 1 in 500 males. It is the most common form of hypogonadism in men (1,6% of infertile men) and occurs due to meiotic non-dysjunction during gametogenesis. Patients present with small firms testes (2-10 ml), gynecomastia, increased height, azoospermia and elevated levels of gonadotrophins. Due to increased estradiol and increased TBG, T levels may be normal although the production is reduced. 10% of the cases are mosaic forms 46 XY/ 47 XXY (due to mitotic non- dysjunction after fertilization of the zygote). Sometimes the mosaicism can be present only in the testes. Azoospermia is present only in 50% and some patients can be fertile (Okada, Hum Reprod 14:946,1999).

  • Microdeletion of the Y chromosome (at the level of a gene controlling spermatogenesis, called DAZ (deleted in azoospermia) in the AZF locus (azoospermic factor), located close to Yq11.23 A microdeletion is detected in 13% of azoospermic men and in 1-7% of severely oligospermic men (Oliva, F St 70:506,1998; Silber 13:3332,1998). Deletions of AZF appear to arise de novo in the general populaton at a rate of 1/10000 male newborns (Johnson F St 70:397,1998; Jiang, F St 71:10291999).
  • XX male syndrome: 1/20000 male births; due to interchange of a Y chromosome gene with the X chromosome (?). Clinically cf Klinefelter. Hypospadias common.(Scheiker 1982)
  • other karyotype abnormalities:
    • Chromosomal abnormalities in somatic cells:
      • trisomy 21 or 8XYY : fertile or spermatogenic arrest.

Chromosomal abnormalities in germ cells: meiotic mutations occur in 4 to 10% of infertile men (anomalies in zygotene and pachyten with abnormal synaptonemal complex) (Johnson, F St 70:312,1998;  Vendrell, Hum Rep 14:375,1999)

3. Sertoli cell only` syndrome:

It accounts for 1/10 to 1/3 of azoospermic patients. Histological findings are characterised by a complete absence of germinal elements occuring in patients with a normal male phenotype and normal caryotype. It can be the result from several etiologies: viral orchitis, cryptorchidism, androgen resistance, familial syndrome. FSH values are usually high, sometimes normal (Turek, F St 64 :1197,1995) Y microdeletions can be detected in 50% of cases (Foresta, Hum Rep 13:302,1998).

4. Spermatogenetic arrest:

This is observed in 4 to 30% of azoospermic patients. Interruption of germ cell differentiation resulting in oligospermia (partial arrest ) or azoospermia (complete arrest). It generally occurs in normal patients with normal testicular volume and gonadotropin levels. Most cases are due to genetic abnormalities occurring in the prophase of the first meiotic division (zygoten and pachyten phase). Acquired cases can be due to hormonal, thermic or toxic factors (Martin-Du Pan, Campana F  St 60:937,1993).

5. Anomalies of sperm structure:

- Immotile cilia syndrome:

Inherited as an autosomal recessive trait. Results in chronic sinusitis and bronchiectasis. The Kartagener syndrome is associated with situs inversus. Due to missing or very short dynein arms, missing central tubules or displacement of one of the nine doublets. In normal subjects 10% of sperms have an incorrect number of microt. doublets and 7% have fewer than 9 doublets (Wilton, JCI 75:825,1985). Asthenospermia can also be due to mid-pieces abnormalities of mitochondria and to deficiency in protein carboxyl methylase (Gagnon, NEJM 306:821,1982). Among 400 patients with absent sperm motility 3% had dynein arm deficiency and 23% were necrospermic (Ryder, F St 53:556,1990).

- Necrospermia: can be distinguished from immotile sperm. syndrome by supravital dyes. Degenerative changes involve all sperm components (Zamboni, F St 48:711,1987)

- Teratospermia: agenesis of the acrosome results in round headed spermatozoa. Familial, polygenic mode of inheritance.Monomorphic round head teratozoospermia are probably of genetic origin whereas testicular factors could be responsible for the amorphous head (Marchini, Andrologia 5:468, 1989). Teratospermia has been associated with autosomal translocations (Warter 81).


The incidence of primary endocrine defects in infertile men is less than 2%

1. Hypogonadotrophic hypogonadism (HH)

Patients with HH show decreased levels of gonadotrophins and T. If it occurs before puberty, signs of eunuchoidism are present: arm span 5 cm greater than height, decreased hair and muscular development, infantile genitalia. If anosmia is present: Kallmann syndrome (frequency:1/10000 to 1 /60000). Absence of neurons secreting GnRH. X linked inheritance or autosomal-dominant or recessive (role of KALIG 1 locus on short arm of chromosome X which encodes for a protein that could be responsible for neuronal migration (GnRH neurons arise in olfactory placode and migrate along the cranial nerve I to the preoptic area)(Bick, NEJM 326 :1752,1992).

Acquired form of HH occurs in patients with normal pubertal developpement with a recent history of decreased sexual function and fertility. Partial defect in gonadotrophin secretion ( low LH and normal FSH) can lead to fertile eunuch syndrome. Hemochromatose must be ruled out by dosage of ferritine.

HH can also be due to pituitary lesions (prolactinoma, Cushing disease) or infiltrative diseases and a IRM of the pituitary is always indicated in case of HH, as well as a dosage of prolactin in case of impotence (cf infra). These conditions can be treated with HCG (3 x 2000 U and HMG 3 x 75-150 U /week) or by pulsatile GnRH if fertility is desired (Buchter, Eur J Endo 139:298,1998).Cryptorchism has a bad prognosis (Finkel, NEJM 313:651,1985).

2. Hyperprolactinaemia

Micro- or macroadenoma of the pituitary secreting prolactin can induce hypogonadism either by impairing GnRH release or by destruction of the pituitary (Segal, 27:1425,1976). It causes loss of libido, visual abnormalities and galactorrhea in 15-30% of cases. Fertility and potency can be recovered after surgical or medical treatment (bromocriptine) .

3. Congenital adrenal hyperplasia

In mild forms of   21-hydroxylase deficiency  high ACTH levels stimulate the synthesis of androgenic steroids by the adrenal cortex (androstenedione and 17 OH P) resulting in precocious puberty and abnormal phallic enlargement. Gonadotrophins are suppressed resulting in some cases in oligospermia. Fertility can be restored by glucocorticoid treatment (Bonaccorsi, F St 47:664,1987).

4. Androgen resistance syndrome

Quantitative or qualitative defects of testosterone binding to the androgen receptor due to mutations in the receptor result in a spectrum of disease ranging from complete testicular feminisation to infertile male syndrome (Griffin, NEJM 326:611,1992). Androgen receptor deficiency has been observed in 0 to 19% of men with idiopathic oligospermia and normal phenotype (Morrow, JCEM 64:1115,1987; Bouchar, JCEM 63:1242,1986; van Roijen, J Androl 16:510,1995). LH levels are slightly increased and T levels are normal. The LH (UI) x Testo (ng/ml) product is increased above 200, but could be normal (100) in some cases (Morrow, JCEM 64:1115,1987). Increased LH x T products have also been reported in coeliac disease (Farthing Gut 23:608; and 24:127,1983) and in hyperthyroidism (with increased estradiol levels in the latter) (Jidd, JCEM 48:798,1979).

The androgen receptor has been shown to contain trinucleotid repeat loci. An increased length of these repeats has been associated with androgen resistance and defective spermatogenesis (Thein, JCEM 82:3777,1997).

5. Slow pulsing oligospermia:

Reduced LH pulse frequency has been observed in oligospermic patients with high FSH levels, which were decreased by GnRH pulsatile administration. However, there is no sperm improvement after GnRH treatment( Bals-Pratsch, Clin Endocrin 30:549,1989). Aromatase inhibitors could improve the sperm count (Scaglia, Hum Rep13:2782,1998)

Nb : Mutation of the FSH receptor does not play a pathogenic role in male infertility (JCEM 84:751,1999)

V. SYSTEMIC DISEASES (Review:Turner 47 :379-403,1997)

1.Renal failure

Renal failure leads to decreased T levels and increased gonadotrophins and prolactin in 25% of cases. Improvement of sperm after zinc administration (Mahajan, Ann Int Med 97:357,1982). Fertility can be restored by kidney transplantation but not by dialysis (Handelsman, Endocr Rev 6:151,1985).

2. Cirrhosis of the liver

Gynaecomastia and impotence are present in half of the cases,estradiol and TBG are increased. Gonadotrophins are slightly increased. Testicular atrophy and hypospermatogenesis are described (Van Thiel, Gastroenterol 67:1188,1975).

3. Sickle cell anaemia:

Testicular atrophy occurs in 1/3, maturation arrest of sperms.  Hypoxaemia and zinc deficiency might play a role (Prasad, Am J Hematol 10:119,1981).

4. Gastrointestinal diseases:

In coeliac disease, a decreased mobility and teratospermy has been observed as well as an increased T x LH ratio . In Crohn's disease oligospermia has been observed in 6/13 patients (Farthing Gut 23 :608,82 and 24 :127,83)

5. Hodgkin disease:

Asthenoteratospermia has been observed in 50% of the cases, oligospermia in 25% (35 patients) and low T levels (Ragni 1985).

6. Neurological diseases :

- myotonic dystrophy: small testes, low T and high gonadotrophins

- spinal cord lesions: moderate oligospermia and most of the time asthenospermia is observed in paraplegics. Multifactorial causes: retrograde ejaculation, urinary tract infection. Not due to hormonal problem or infrequent ejaculations. Semen collection by rectal probe electrostimulation or vibrator (Perkash, J Urol 134:284,1985).

7. Psychological factors:

Infertile men do not present a special psychopathological profile and compared to a control group of fertile men they do not present differences in personality profile and coping strategies (Golombok, Hum Rep 7:208,1992). No sperm alteration has been observed in male marathon runners and in depressed patients (Jensen, F St 64:1189,1995; Amsterdam Psychosom Med 43:183,1981). However, the stress due to IVF procedure and the stress due to the loss of a close parent or due to earthquake but not the stress at work could decrease slightly semen quality (Hum Rep 14:753,1999 and 11:1244,1996; Fenster, J Androl 18:194,1997).


Genital duct obstruction is found in 5 to 7% of infertile patients. Obstruction may occur at any level of the genital tract. It can be congenital or acquired, secondary to infection (bilateral epididymitis), stricture or vasectomy. Most of the patients present with azoospermia, normal size testes and normal gonadotrophin levels. In congenital absence of the vas there is usually an associated absence of the seminal vesicles and ampulla. The semen volume is low, acid and fructose negative.

Incomplete or unilateral obstruction of the male genital tract can be responsible for oligospermia and is associated with circulating antisperm antibodies (Hendry, Hum Rep 9:463,1994; Belmonte, Hum Rep 13:3402,1998). A trial with an anti-inflammatory treatment such as diclofenac could be useful (Martin-Du Pan, Hum Rep 12:396,1997)

Investigations and treatment will be discussed by Dr de Boccard


1. Retrograde ejaculation:

It can be suspected in case of "dry" ejaculation or small volume of the ejaculate. It may follow transurethral resection of the prostate, bladder neck surgery, retroperitoneal lymph node dissection or pelvic surgery (rectum). It occurs in diabetes with peripheral neuropathy, multiple sclerosis, paraplegia and alpha-adrenolytic drugs. Sperm can be recovered in the urine after alkalinisation (650 mg of bicarbonate 4 x /d 48 h prior to collection). Imipramine (25-50 mg/d) can be tried to re-establish antegrade ejaculation (Int J Androl 22:173,1999).

2. Ejaculation failure :

A complete absence of antegrade ejaculation can be due to sympathetic denervation, autonomic medications or psychogenic problems. Retarded ejaculation can be a milder form of this condition. Treatment includes vibratory stimulation, electroejaculation and psychotherapy (Urol Clinics of North America 14:583,1987).


Impotence and premature ejaculations. Discussed by Prof. Ruedi.


In 30 to 50% of cases (if we include cases with no sperm improvement after varicocele repair or prostatitis treatment) no aetiology can be identified to explain abnormal semen or infertility. Abnormalities of all semen parameters are usually observed. Slight increase of FSH values may result from injury of the testis due to viral, toxic or congenital factor. In 1/3 of the cases of idiopathic infertility with apparently normal sperm, there is a decreased rate or a lack of oocyte fertilisation with IVF. A decreased binding to zona pellucida has been observed in 28% of cases and an absence of sperm hyperactivation induced by follicular fluid in another 39% (from 18 patients). In these patients (with idiopathic infertility), reactive oxygen species generation was not different from the control group (semen samples producing high rate of free oxygen radicals are characterised by a loss of sperm function) ( Mc Kenna, F St 59:405, 1993). Ultrastructural defects of sperm head or tail or defects of acrosine reaction could also be responsible for some cases of lack of IVF (Zamboni, F St 48:711,1992). Immunological factors could be responsible for another 10% of cases ( by decreasing sperm binding to the zona pellucida). Androgen receptor deficiency is underdiagnosed although the prevalence rate is much lower than the 40% rate observed by Aiman (JCEM 54:725,1982). Genetic diseases are also underdiagnosed in case of chromosomal anomalies present exclusively in germ cells (Vendrell, Hum Rep14:375,1999).