Application of seminal germ cell morphology and semen biochemistry in the diagnosis and management of azoospermic subjects

Somnath Roy^1,2, A. Banerjee¹, H.C. Pandey¹,  G. Singh¹, G.L. Kumari²

¹National Institute of  Health and Family Welfare (NIHFW),  New Delhi-110067, India 
²Reproductive Health Foundation (RHF), New Delhi, India

Asian J Androl  2001 Mar; 3: 55-62

Keywords: zoospermia; semen; biochemistry; germ cells; intracytoplasmic sperm injection

Abstract

Aim: To evaluate whether the study of seminal germ cell morphology (SGCM) and semen biochemistry could be fruitfully utilized for the diagnosis and management of azoospermic subjects. Methods: In the semen, mature and immature germ cells are contributed by the testes, 70% of glycerylphosphoryl choline (GPC) by the epididymis, fructose mostly or solely by the seminal vesicles and acid phosphate (ACP) by the prostate.  In 16 normal volunteers, 12 vasectomized subjects and 186 azoospermic subjects, these parameters have been studied and the data have been analyzed. Results: Both mature and immature germ cells are absent in the semen of vasectomized subjects as well as in obstructive azoospermia; GPC level is also significantly decreased in both these groups. In cases with non-obstructive azoospermia immature germ cells are present and seminal GPC, ACP and fructose levels are normal.  The diagnosis of obstructive and non-obstructive azoospermiabased on these parameters correlated well with correct testicular biopsy findings.  In some cases of azoospermia due to hypospermatogenesis or spermatogenic developmental arrest, the SGCM studies were very helpful in objectively monitoring the response of the germinal tissue to specific treatments. Conclusion: SGCM and semen biochemical parameters are very valuable non-invasive markers for differentiating obstructive from non-obstructive azoospermia.  The SGCM findings serve as a dependable non-invasive testicular marker with high predictive value.

1 Introduction

A significant proportion of infertile male subjects present with azoospermia.  Some of them might have had varying degrees of oligospermia prior to development of azoospermia.  For elucidating the real nature and etiology of such disorders, a systematic approach and proper insight are needed.  Firstly, it is to be determined whether the azoospermia is due to (a) an obstructive  cause, or  (b)a non-obstructive cause; then it would be important to carry out other specific tests to find out its possible etiology^[1]. 

During mid-1970s and early 1980s we were engaged in studies with cyproterone acetate (CPA) a progestogen with specific anti-androgenic property, with or without testosterone enanthate (TE) as a potential male contraceptive.  In order to elucidate the nature, site and mode of action of these regimens we studied among other parameters, seminal germ cell morphology (SGCM) and semen bio-chemist ry: glycerylphosphorylcholine (GPC), acid phosphatase (ACP) and fructose^[2,3]. The use of CPA alone or in combination with TE caused spermatogenic suppression. The sperm concentration in the semen gradually decreased and in many azoospermia was produced. During   this decline in sperm count, there was a gradual increase in percentage of immature germ cells which could be seen in the semen even in azoospermic state. This observation gave us the idea that the presence of immature germ cells in the semen of azoospermic subjects could be used as a dependable testicular marker of germinal cell origin.

2 Materials and methods

At the initial stage, a group of normal volunteers and some vasectomized subjects were included in the study along with azoospermic patients for comparative analysis of the findings. Data on 16 normal volunteers, 12 vasectomized subjects and 186 azoospermic subjects have been analyzed.  Cases of retrograde ejaculation were excluded after appropriate diagnostic tests.

In  all the subjects, besides an adequate history, a thorough  physical examination was carried out.  Particular attention was given to:  (a)  the volume and consistency of the testes;  (b) the size, feel, nodularity and tenderness of the epididymides;  (c) the presence or absence of vasa deferentia, and  (d) the presence of any varicocele.  Routine physical and microscopical examinations of  the semen were performed, and special seminal studies included germ cell morphology and semen biochemistry comprising GPC, ACP and fructose. These studies  were carried out as described in our earlier publications^[2,3] and in the WHO  Manual^[4].

The condition of azoospermia may often have associated  with infection  and/or  inflammation of the secondary sex organs such as the epididymides and seminal vesicles, which might cause temporary blockage of the ducts.  Keeping this in mind, after a critical review of the physical findings of the genitalia and the preliminary seminal picture, some of the azoospermic subjects were administered a course of antibiotics plus an anti-inflammatory drug with or without doing a prior semen culture.  Then they were subjected to studies of SGCM and semen biochemistry. 

Semen samples were collected after 3 to 4 days of abstinence; sometimes 2 to 3  samples had to be pooled because of low volumes.  After centrifugation the deposit was properly smeared on glass slides and appropriately stained.  The immature germ cells at various stages of differentiation, i.e., spermatogonia, primary and secondary spermatocytes, and round and elongated spermatids were identified.  The round spermatids need to be differentiated from lymphocytes, and also from the nuclei of Sertoli cells, which have characteristic nucleolus.  Sometime the round spermatids share common cytoplasm and may appear as polymophonuclear leucocytes.  Great care is needed to differentiate the germ cells from the leukocytes by the nuclear size and staining characteristics. 

3 Results

Over the years, these tests have been performed routinely on several hundred subjects, and these have provided very dependable results in differentiating obstructive from non-obstructive azoospermia.  The summary findings have been incorporated in one of our earlier publications^[5].  Data on a total of 214 subjects are analyzed for incorporation into   this presentation. The salient differentiating features of various categories of subjects are summarized in  Table 1.  

Table 1.  Mean testicular volume, and seminal germ cells and biochemical constituents in different categories of subjects.

Legend: Mature and immature germ cells are absent in the semen of vasectomized subjects as well as in obstructive azoospermia; GPC is also significantly decreased in these cases.  In cases with non-obstructive azoospermia immature germ cells are present and seminal GPC as well as ACP and fructose are normal.  It is to be noted that in some cases of obstructive azoospermia testes volume may be decreased, and conversely in some cases of non-obstructive azoospermia testes volume may be normal.

It may be noted from Table 1 that in obstructive azoospermia the level of GPC is low and there is no mature or immature germ cells in  the semen, as are observed in vasectomized individuals.  The level of fructose and ACP activity are not altered in vasectomy or obstructive azoospermia, except in rare cases with non-development or mal-development of seminal vesicles where seminal fructose may be absent or very low.  In cases with obstruction at common ejaculatory ducts, the semen volume is very low and is acidic, fructose is absent, GPC is low and ACP concentration is high. The data on these  rare cases are not presented in this report. 

Table 2.  Correlation of the findings of testicular biopsy with seminal germ cells and GPC in subjects with obstructive and non-obstructive azoospermia.

Legend: Of  the 70 testicular biopsies studied, 66 belonged to cases with non-obstructive azoospermia and 4 to obstructive azoospermia.  Abnormal spermatogenesis was observed in all 66 subjects with non-obstructive azoospermia, and spermatogenic maturation arrest was noted in two cases with obstructive azoospermia.  In all the four cases of Sertoli-cell-only syndrome immature germ cells were found in SGCM study.

4 Discussion

Azoospermia can either be excretory (obstructive) or secretory (non-obstructive). The latter is usually associated with a reduced testicular volume and an elevated blood level of FSH.  Therefore, FSH has long been considered to be a reliable marker of the status of spermatogenesis; elevated FSH represents hypospermatogenesis and azoospermia^[6]  and  normal FSH is associated with obstructive azoospermia. However, recent studies with increasing use of testicular  screening have clearly demonstrated that plasma FSH can no longer be considered to be definitive marker for azoospermia^[7-9]. Therefore, there is a need for development and use of more dependable seminal markers that could consistently discriminate between obstructive and non-obstructive azoospermia.  It has been felt that such markers must be of testicular origin, disappear after vasectomy, be specifically secreted by the Sertoli or germinal cells at high concentrations, be associated with spermatogenesis and be still present in detectable concentration in seminal plasma in cases with hypospermatogenesis^[10].  Several such markers have been tried, e.g. lactate dehydrogenase (LDH-X)^[11], transferrin^[12], insulin-like growth factor and ₂ macroglobulin^[13] and inhibin^[14], but none appears to be a convenient testicular marker either for diagnosis or prognosis.

More recently, the potential use of seminal concentration of anti-Mullerian hormone (AMH) has been suggested^[10]. AMH is a glycoprotein and is specifically secreted in the male by the Sertoli cells.  Its major function which has been identified so far is that it causes regression of Mullerian duct elements in the male fetus.  Its blood level falls dramatically at puberty, but it is found in high concentration in the seminal plasma of fertile men^[15,16]. Very little is known about the function of AMH in post-natal life, but there is some evidence to indicate that it may control proliferation and steroidogenic function of Leydig cells^[17] and that it may be related to germinal cell proliferation^[18].  Fnichel et al^[10] reported that AMH was present in the seminal plasma of most fertile donors, was undetectable in all subjects with obstructive azoospermia and  was low in patients with non-obstructive azoospermia.  It has been suggested by these authors that seminal AMH may represent a non-invasive marker that would not only differentiate between obstructive and non-obstructive azoospermia, but its presence at low level may be a predictive indicator of persistent hypospermatogenesis in non-obstructive azoospermia.  However, at the current state of development, seminal AMH secreted by the Sertoli cells can not be used as a dependable marker as it was undetectable in some fertile subjects and its relation or inference in regard to the status of spermatogenesis is at best indirect.  Its marked decrease noted in association with spermatogenic failure may actually reflect a primary alteration in Sertoli cell function; that also would lead to spermatogenic arrest.

With the object of making a differential diagnosis between obstructive and non-obstructive azoospermia, testicular biopsy or FNAC is often performed. Problems are sometimes encountered in interpreting testicular biopsy of azoospermic individuals.  In our study, in nine subjects the testicular biopsy done elsewhere was reported to have mature  sperm, indicating an obstructive cause for azoospermia.  Based  on   this report, the urologist advised surgical exploration and vaso-epididymal anastomosis.  One actually got operated, but azoospermia continued; the other eight refused to have surgery.  In all of them our SGCM study showed immature germ cells, implying a non-obstructive cause for azoospermia.  It should be recognized that sometimes the testicular biopsy procedure and the fixing  and processing of the tissue may not be proper, or despite being properly prepared, it may not be interpreted appropriately.  As for example, even in well equipped centres the advanced stages of  spermatids (Scd) which may appear as tail-less spermatozoa, may be interpreted as sperm; and such testis biopsy has been wrongly diagnosed as having normal spermatogenesis^[19]. Consequently, based on this wrong interpretation vaso-epididymal anastomosis may be attempted.  However, in reality, this may be a case of non-obstructive azoospermia due to spermatogenic development arrest, as demonstrated above.

We had four subjects presenting with azoospermia whose testicular FNAC was studied elsewhere before being seen by us. In three of them sperm were reportedly seen in FNAC and a diagnosis of obstructive azoospermia was made, and they were advised surgical exploration and vaso-epididymal anastomosis.  In our SGCM study, all these three subjects had immature germ cells in the semen, thus establishing the diagnosis of non-obstructive azoospermia.  In one of these cases the senior author had the opportunity of reviewing the FNAC slide.  This interesting case report will be illustrated in some detail shortly.  In the fourth case, a diagnosis of spermatogenic maturation arrest was made from FNAC, and our SGCM  study also confirmed this.

The values of SGCM study in the differential diagnosis of obstructive vs non-obstructive azoospermia are well evident from the above data.  However, their utility and  importance will be further  illustrated by the four  case reports that will be presented later on; these include three additional subjects.

Earlier, in the management of patients presenting with azoospermia there was very little scope for achieving pregnancy.  In some selected cases of obstructive azoospermia microsurgical correction was feasible, and for cases with non-obstructive azoospermia there was no hope.  However, with the recent development of intracytoplasmic sperm injection (ICSI) technique, which allows fertilization with very few spermatozoa, extraordinary advances have been made.  In case of obstructive azoospermia, epididymal puncture or testicular biopsy can yield sufficient number of sperm for ICSI; and in majority of subjects with testicular failure caused either by maturation arrest, Stertoli-cell-only syndrome, cryptorchid testicular atrophy or even Klinefelter's syndrome, there are very tiny number of spermatozoa or spermatids which could be extracted from extensive testicular biopsies and utilized for ICSI^[20-23]. Recent reports have further suggested that recovery of spermatozoa from the testis may be possible in more than 50% of cases of non-obstructive azoospermia regardless of clinical parameters concerning the size of testes and FSH levels in the blood^[24]. Even lack of spermatozoa in one testicular biopsy does not confirm complete lack of spermatozoa in the testes^[25]. In about 43% cases of non-obstructive azoospermia, repetitive multiple biopsies enabled recovery of sufficient sperm for ICSI, despite a negative preliminary biopsy;   this indicates the presence of focal hypospermatogenesis^[24]. However, repetitive surgery has psychological, financial and potential physical implications.  Unnecessary/unfruitful repeated biopsies should be avoided.  Therefore, dependable non-invasive markers are urgently needed to indicate whether biopsies should be repeated or not. Our studies have demonstrated that the seminal germ cells (SGCs) could  serve as such marker.  The following case reports of four azoospermic subjects would further illustrate the value of  SGCM.

Case 1: A  27 year old subject was investigated for azoospermia in another centre.  Testes were reportedly small.  FNAC  showed spermatogenic cells at all stages and a few mature sperm were also seen.  The pathologist's impression was normal spermatogenesis in both testes, and the inference was obstructive azoospermia.  Subsequently,   this patient was investigated by us.  The testes were small (5 mL and 3 mL).  The SGCM study done twice failed to show any sperm and immature cells, and only up to secondary spermatocytes were seen.  Seminal GPC, ACP and fructose, estimated twice, were all in normal range.  The blood level of FSH was normal, LH and testosterone were borderline low, and prolactin (PRL) was borderline high.  The FNAC slide was reassessed by the senior author, and he confirmed the presence of spermatogenesis and mature sperm.  What could be the possible reason for the conflicting findings of the FNAC and SGCM?  It was argued that there was no obstruction in the passage; the testes were very small and whatever small number of sperms were being formed these could not reach the ejaculate intact.  If that  interpretation was correct, with proper stimulation of spermatogenesis sperm should appear in the semen.  With this idea the patient was treated with hCG 2000 IU injected twice weekly for three months. The testes were enlarged to 7 mL and 5 mL sizes, respectively;  SGCM  showed progression to late stage spermatids and a few spermatozoa. This confirmed our diagnosis of non-obstructive azoospermia due to severe hypospermatogenesis, which could be stimulated with hormonal treatment and sperm appeared in the semen.

Case  2:  In  a 39 year  old subject, repeated semen analysis during 1984-1990 reported  azoospermia except  two tests in 1984 and 1988 when 1 or 2 sperm and 0-3  sperms/hPF were noted, respectively.  During 1987 & 1988 he was treated unsuccessfully by two urologists in Delhi. Ultimately, he was investigated by a consultant urologist in UK, who did bilateral scrotal exploration and vasogram. From a detailed note provided by this consultant the following relevant excerpts are reproduced here: Left side ...........impossible to perform anastomosis on   this side.  Right side .............obstruction is at higher level than epididymis.  With the present state of knowledge nothing can be offered for remedy.

Then the couple decided to go to USA for IVF & ET with donor sperm.  But upon the advice of a friend he came to consult us.  On examination,  the testis volume was 18 mL and 15 mL and the vas deferens could be palpated properly on one side only.  SGCM  showed immature germ cells up to secondary spermatocytes only, and seminal GPC, ACP and fructose levels were normal.  These findings clearly established that he had non-obstructive azoospermia and there was no obstruction as was diagnosed earlier. Blood levels of FSH, LH and testosterone were normal, but blood prolactin (PRL) level was very high (93 ng/mL, normal range 0-15). He was put on adequate dose of bromocriptine and the response was monitored by the SGCM and blood PRL levels.  With the suppression of PRL, spermatogenesis progressed gradually, and at the end of about seven months plenty motile sperm (2 to 4/HPF)  were  noted in the ejaculate.

Case 3: A 32 year old subject with a history of mumps at the age of 9 yrs had repeated semen tests reporting azoospermia.  Physical examination showed moderate androgenisation and testes size 12 mL & 14 mL. Blood hormones FSH, LH, PRL & TSH were normal; testosterone was at lower border of normal.  Semen GCM showed immature germ cells of all types up to round and elongated spermatids, but no spermatozoa.  Seminal GPC, ACP and fructose were normal. He was diagnosed idiopathic non-obstructive azoospermia.  Initially, he was treated with clomiphene citrate and hCG.  In September 1997, SGCM showed that immature germ cell population was thick and percentages of round  (48%) and elongated (26%) spermatids were increased.  He was put on hMG and hCG. In November 1997, SGCM showed increased percentage of elongated spermatids (66%), but no spermatozoa were seen.  It was decided to increase the dose of hCG.

In January 1998, SGCM showed 4 spermatozoa for the first time. The doses of both hCG and hMG were increased.  SGCM studies were repeated in February, April, June, August and December 1998.  Sperm (7 to 18) were seen in the  ejaculate each time, except in Feb 1998.  In the meantime negotiations were being made with the IVF centres for doing ICSI.  Testicular FNAC was done in September 1998, which showed Stertoli-cells (58%) and the rest comprised all stages of immature germ cells, but no sperm.  Ultimately,  ICSI was performed  in February 1999 using sperm from testicular biopsy,  tissue being obtained from both sides. The material contained plenty of Sertoli-cell nuclei and only few motile sperm were seen under high power scattered in different fields.  A thorough search yielded adequate number of sperm for ICSI.  This resulted in pregnancy and a healthy male baby was born at term.

This case report clearly shows that the study of seminal germ cell morphology (SGCM) and semen biochemistry not only clearly established the diagnosis of non-obstructive azoospermia, but the SGCM was very useful in objectively monitoring the response of germinal cells to specific hormonal stimulation. 

Case   4:  A 30 years old subject had repeated semen tests elsewhere which revealed azoospermia.  Bilateral testicular biopsy was reported to show  tubules predominantly lined by Sertoli cells and only few germinal cells could be seen.  Features suggestive of testicular atrophy.  ICSI was tried twice elsewhere using sperm from testicular  aspiration through needle, but with no success.

In October 1999 he was seen by us. Physical: normal androgenisation,  testes size-18 mL & 17 mL,  semen germ cell morphology-no spermatozoa, immature cell population-thin, spermatogonia-1%, spermatocytes-primary 6% & secondary 20%, spermatids-round 65% & elongated 6%. Seminal GPC, ACP and fructose were within normal limits. Levels of plasma FSH, LH, PRL and total testosterone were within normal limits. However, salivary testosterone, which reflects the free form of testosterone, was low.

The testicular biopsy was reviewed and the findings were: Tubules are lined by Sertoli-cells only; in occasional tubules very few (1 to 5) immature germinal cellsspermatogonia and primary spermatocytes and in very rare ones secondary spermatocytes, were noted. Occasional tubules showed hyalinization. Impression: Sertoli-cell-only syndrome-type II  (i.e. with some germ cells).Since November 1999 he was treated initially with  clomiphene citrate orally and intramuscular injections of depot testosterone, and later with hCG.  In May 2000 he was put on hMG plus hCG three times weekly. On 27 October 2000, SGCM report shows:  immature cell population has become moderately thick and elongated spermatid percentage has increased to 27% (originally 6%).  These indicate multiplication of germinal elements and some progression in spermatogenic maturation process. Further increase in the percentage of elongated spermatids would  indicate high probability of finding and extracting spermatozoa from extensive testicular biopsy.  Thus the SGCM findings provide high predictive value even in a case with Sertoli-cell-only syndrome.

The above cases clearly illustrate the importance of SGCM  and semen biochemistry in differentiating between obstructive and non-obstructive azoospermia and in clinching a correct diagnosis.  SGCM also provides valuable criteria for objectively monitoring the response of germinal cells to specific treatments. It serves as a dependable non-invasive testicular marker with high predictive value even in cases with Sertoli-cell-only syndrome.  In the latter case, the presence of germinal cells may be missed in testicular biopsy,  but the germ cells are conveniently detected by  SGCM study as it scans the exfoliated cells drained from the entire testicular tissue of both sides.

In addition, we have had several patients with azoospermia due to spermatogenic maturation arrest at different cytological levels resulting from hypogonadotropic hypogonadism, hypoandrogenism, hyperprolactinemia, varicocele or idiopathic hypospermatogenesis, where SGCM studies have been very helpful in objectively monitoring the response of the germinal epithelium to specific treatments.  Sperm appeared in their ejaculate, and  some of them impregnated their wives through natural sex act. Others provided the basis for non-invasive as well as invasive approaches of obtaining sperm for performing ICSI  (unpublished observation).

It is clearly evident from the above that the study of SGCM has major advantages over all other testicular markers tried so far, including AMH, as it directly reflects the state of spermatogenesis.

5 Conclusion

In azoospermic subjects, we have studied the seminal germ cell morphology (SGCM)  and semen biochemistry comprising  the levels of GPC, ACP  and fructose, which served as markers of contributions from the testis, epididymis, prostate and seminal vesicles, respectively. These proved to be very valuable non-invasive tools for differentiating between obstructive and non-obstructive azoospermia.  In some cases of azoospermia where testicular biopsy or FNAC done in other centres had failed in establishing a proper diagnosis, our studies with SGCM  and semen biochemistry have clearly clinched the issue.

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Correspondence to: Prof. Somnath Roy, Emeritus Professor and Former Director, National Institute of Health & Family Welfare, Munirka, New Delhi-110067, India.
Tel: +91-11-618 8485, 646 2943    Fax: +91-11-610 1623  
e-mail: nihfw@delnet.ren.nic.in; nihfw@mantraonline.com; sarveshwara@vsnl.com
Received 2000-12-24     Accepted 2001-02-20