TESTICULAR BIOPSIES IN INFERTILITY AND CORRELATION WITH ...repository-tnmgrmu.ac.in/4732/1/200301117priscilla_babu.pdf · 1 CERTIFICATE This is to certify that this dissertation titled

TESTICULAR BIOPSIES IN INFERTILITY AND CORRELATION WITH

CLINICAL AND LABORATORY FEATURES

A dissertation in part fulfillment of the rules and regulations

for the M.D. Branch III (Pathology) Degree Examination of the

Tamil Nadu Dr. M.G.R Medical University, to be held in April

2017.

1

CERTIFICATE

This is to certify that this dissertation titled “Testicular biopsies in infertility and

correlation with clinical and laboratory features” is a bonafide work done by

Dr.Priscilla Babu, in part fulfillment of the rules and regulations for the M.D. Branch

III (Pathology) Degree Examination of the Tamil Nadu Dr. M.G.R Medical University,

to be held in April 2017.

Dr. Vivi M. Srivastava, MBBS, MD

Professor and Head,

Department of General Pathology,

Christian Medical College, Vellore

Dr. Anna Pulimood

Principal,

Christian Medical College,

Vellore.

2

CERTIFICATE


correlation with clinical and laboratory features” is a bonafide work done by

Dr.Priscilla Babu, in part fulfillment of the rules and regulations for the M.D. Branch

III (Pathology) Degree Examination of the Tamil Nadu Dr. M.G.R Medical University,

to be held in April 2017.The candidate has independently reviewed the literature,

performed the data collection, analyzed the methodology and carried out the

evaluation towards completion of the thesis.

Dr. Vivi M. Srivastava, MBBS, MD

Professor and Head,

Department of General Pathology


Vellore.

3

CERTIFICATE


correlation with clinical and laboratory features” is a bonafide work done by me,

under the guidance of Dr. Vivi M Srivastava, in part fulfillment of the rules and

regulations for the M.D. Branch III (Pathology) Degree Examination of the Tamil Nadu

Dr. M.G.R Medical University, to be held in April 2017. I have independently reviewed

the literature, performed the data collection, analyzed the methodology and carried

out the evaluation towards completion of the thesis.

Dr. Priscilla Babu,

Post-graduate registrar,

Department of General Pathology


Vellore.

4

5

List of tables:

Table 1 Age distribution

Table 2 Testicular volume

Table 3 Semen analysis

Table 4 Hormonal profile

Table 5 Distribution of patients according to FSH levels.

Table 6 Classification based on number of seminferous tubules in the biopsy

Table 7 Tubule diameter and basement membrane thickness

Table 8 Features of interstitium

Table 9 Histology of seminferous tubules

Table 10 Profile of patients with hypospermatogenesis

Table 11 Stages of maturation arrest

Table 12 Profile of patients with maturation arrest

Table 13 Profile of patients with Sertoli cell only

Table 14 Profile of patients with peritubular fibrosis and atrophy

Table 15 Comparison of testicular volumes across the various histological profiles

Table 16 FSH variation across various histological profiles.

Table 17 Classification based on karyotype done on 24 patients.

Table 18 Classification based on Y chromosome microdeletion status

Table 19 Relation of Histology to karyotype and Y chromosome mcrodeletion in

the abnormal cases

Table 20 Testicular biopsies in infertile males: Comparison of the histological

patterns in our patients and those from other countries

Table 21 Testicular biopsies in infertile males: Comparison of thehistological

patterns in our patients and those from India

Table 22 Histological profile- change in pattern over the years in Saudi Arabia.

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List of figures:

Fig.1 Stages of spermatogenesis

Fig.2 Stages of spermatogenesis within the seminiferous tubule

Fig.3 Histology of testis

Fig.4 Cross section of seminiferous tubule

Fig.5 Paracrine control of spermatogenesis

Fig.6 Sertoli cells only

Fig.7 Maturation arrest

Fig.8 Hypospermatogenesis

Fig.9 Atrophy and peritubular fibrosis

Fig.10 Mixed pattern showing Sertoli cell only pattern in one tubule with

normal spermatogenesis in the background

Fig.11 Algorithm for evaluation of testicular biopsy in infertility

Fig.12 Schematic representation of Y chromosome

Fig.13 Representation of the cytological bands of the Y chromosome.

Fig.14 Granulomatous inflammation

Fig.15 Normal distribution of Leydig cells and lymphocytes

Fig.16 Leydig cell clusters

Fig.17 Leydig cell hyperplasia with peritubular fibrosis

Fig.18 Leydig cell hyperplasia

Fig.19 Normal spermatogenesis, 20X

Fig.20 Normal spermatogenesis, 40X

Fig.21 Hypopermatogenesis, showing occasional mature spermatozoa

Fig.22 Maturation arrest at the level of spermatogonia

Fig.23 Maturation arrest at the level of primary spermatocytes

Fig.24 Maturation arrest at the level of secondary spermatocytes

Fig.25 Sertoli cells only

Fig.26 Atrophy of seminiferous tubules

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Fig.27 Peritubular fibrosis

Fig.28 Statistical correlation between tubule diameter and histology

Fig.29 Karyotype of 45,X male, with SRY gene insertion on chromosome 1.

Fig.30 FISH probe showing absence of signal for Y chromosome.

Fig.31 FISH probe showing insertion of SRY gene at long arm of chromosome 1.

Fig. 32 Y chromosome microdeletion study showing deletion of the AZF b

Fig.33 Y chromosome microdeletion in AZF a, b & c regions, in 45, X male.

Fig.34 Bar diagram showing the most prominent histological category in each

study as compared to the current study.

Fig.35 Bar diagram showing most prominent histologic category in each Indian

study.

Fig.36 Bar diagram showing the shift in histology over the years in Saudi

Arabia

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TABLE OF CONTENTS

INTRODUCTION 1

AIMS AND OBJECTIVES 3

REVIEW OF LITERATURE 4

PATIENTS AND METHODS 44

RESULTS 48

DISCUSSION 77

CONCLUSIONS 87

LIMITATIONS 88

BIBLIOGRAPHY 89

APPENDIX 97

1

INTRODUCTION:

Infertility is a commonly encountered problem in approximately 15% of couples who

wish to conceive. The causes are varied and can be divided as endogenous and

exogenous with respect to testicular origin. The pre-testicular causes are

predominantly hormone associated, at the level of the hypothalamus, pituitary or the

adrenals. The testicular causes are related to the morphology and structural aspects of

the testes. The post-testicular causes are obstructive in nature, and are related to

normal testicular status.

It has been observed that there is a progressive decline in the number of testicular

biopsies performed, in centres all over the world and in our country. The work- up of a

patient seeking help for infertility involves recording of the history and clinical

findings and an array of laboratory tests. Physical parameters such as BMI (body mass

index), secondary sexual characteristics, including testicular volume are assessed to

exclude Klinefelter syndrome or other disorders of sexual development. Laboratory

investigations are asked for. These include a semen analysis and a hormonal profile to

estimate the levels of Follicle Stimulating Hormone (FSH), Luteinizing hormone

(LH), Testosterone and Prolactin. Although most centres perform the entire hormone

panel, it may not be feasible in all centres. If the semen analysis shows azoospermia, it

is essential to determine whether the cause is primarily testicular failure or merely due

to blockage in the passage of otherwise normal, motile sperms. A needle aspiration

technique known as testicular sperm aspiration (TESA) is performed to retrieve

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sperms provided the testicular volume is adequate. The advantages of TESA are that

this procedure does not require expertise and can, therefore, be done even by

internists. However, for the same reason, it must be remembered that the yield of

sperms may be poor. If sperms are not obtained by TESA, or if the testicular volume

is low, either a procedure called micro-dissection testicular sperm extraction

(microTESE) or a testicular biopsy is done. In microTESE, the testicular tissue is

examined at 25X and the presence or absence of sperms confirmed. MicroTESE or

testicular biopsy is attempted depending upon the facilities available. MicroTESE has

the advantage that small amount of tissue can be surgically removed with precision

ensuring a better yield of sperms and better preservation of testicular function, also,

facilitating freezing this tissue for assisted reproductive techniques(ART). Testicular

biopsy provides a larger amount of tissue for examination but cannot be preserved for

use in ART. These procedures are also done to determine the condition of the

testicular tubules in non-obstructive azoospermia, when the presence of sperms is not

guaranteed. With the options of TESA which is less invasive and microTESE, the

number of testicular biopsies being done for infertility has declined. However, the

importance of histological assessment lies in providing a more accurate picture of

testicular pathology and identifying conditions such as hypospermatogenesis, which

may not be evident on microTESE, or Intratubular Germ Cell Neoplasm (ITGCN)

which may be an incidental finding. The importance of an accurate histologic

assessment lies in tailoring treatment and further management and counseling

accordingly. Depending on the presence of mature sperms in the biopsy, further sperm

extraction can be planned.(1)

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AIM: To study the histopathology of testicular tissue in men with azoospermia, and

correlate the findings with clinical features, serum levels of sex hormones and

karyotype and Y chromosome micro-deletion status when available.

OBJECTIVES:

i. To describe the histological features of testicular tissue in males being

evaluated for infertility with special reference to the status of the

germinal epithelium (all stages of maturation seen/ maturation arrest,

including stage of arrest/ oligo- or azoospermia/ Sertoli cells only/

hyalinised testis), and the interstitium (quantitation of Leydig cells/

presence of inflammatory infiltrate/ status of vasculature)

ii. To correlate the histology with the clinical features and biochemical

features namely, serum levels of the following sex hormones in these

patients: luteinizing hormone (LH), follicle-stimulating hormone (FSH),

prolactin (PRL) and testosterone.

iii. To review the karyotype and Y chromosome microdeletion status of

these patients, if available

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REVIEW OF LITERATURE:

Infertility is best defined as the inability to conceive after one year of unprotected

intercourse, and could be due to factors in both males as well females. Male factors

are identified in almost 50% of infertile couples who seek medical intervention.(2)

Causes of infertility in men include

infections such as mumps, tuberculosis, syphilis;

varicocoele (caused by various mechanisms including testicular hypoxia,

venous hypertension, elevated temperature, increase in spermatic vein

catecholamines, and increased oxidative stress);

obstruction of the spermatic ducts;

agglutination of sperm (owing to the presence of anti-sperm antibodies), high

semen viscosity;

necrospermia (dead sperms due to most of the infections and inflammation,

decrease in prostatic fluid leading to non-liquefaction of semen, excessive

amount of sperm and poor sperm motility);

low volume of ejaculate (due to retrograde ejaculation or anejaculation);

ejaculatory dysfunction;

high sperm density;

idiopathic, when no cause can be identified. (3)

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SPERMATOGENESIS:

Spermatogenesis is the sequence of developmental events by which spermatogonial

stem cells give rise to functional spermatozoa. Process of spermatogenesis starts at

puberty and continues throughout life. Trillions of sperms are produced throughout

life.

This process takes place in the seminiferous tubules of the male testis. In the

mammalian testis, spermatogonia arise from primordial germ cells, which migrate into

the developing testis during fetal life. Here, they become associated with the

differentiating Sertoli cells and seminiferous cords are formed. In this setting,

primordial germ cells transform into gonocytes, which remain centrally placed,

surrounded by the immature Sertoli cells. Following a period of multiplication, the

gonocytes migrate adjacent to the basement membrane of the seminiferous tubule

where they divide to form type A spermatogonia. (4,5)

Spermatogenesis can be divided into three stages:

• Spermatogonial stage – In this stage the spermatogonia undergo mitosis to form two

types of cells, type A which replenish the pool of spermatogonia, and type B or

primary spermatocytes which undergo meiosis to produce four haploid gametes.

• Meiotic stage –In this stage each primary spermatocyte divides by meiosis into two

haploid secondary spermatocytes which then give rise to four spermatids

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• Spermiogenesis stage – In this stage the spermatids differentiate into mature

spermatozoa by developing morphological changes of spermatids into spermatozoa

Fig1: Stages of spermatogenesis(6)

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Fig 2: Stages of spermatogenesis within the seminiferous tubule (6)

Each of these stages is discussed below

Spermatogonial stage

The first phase of spermatogenesis begins with the division of the spermatogonia that

line the seminiferous tubule near the basement membrane. Type A spermatogonia

divide to maintain the population of the stem cell pool. Some spermatogonia resulting

from these mitotic divisions stay in the resting pool and do not differentiate, while the

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remaining type A spermatogonia proliferate and undergo several stages of division

and differentiation. The number of spermatogonial cell types identified varies between

species. In the human, type A (dark) and type B (pale) spermatogonial cells can be

distinguished. In the rodent testis, multiple type A spermatogonia, intermediate and

type B spermatogonia have been identified.(4) The type B spermaogonia undergo

mitosis to form the primary spermatocytes.

Meiosis

The primary spermatocytes enter the first cycle of meiosis, from which they

immediately go into the S phase, or the preloptotene phase. During this phase, their

DNA content doubles and they undergo a change in location to inhabit the adluminal

compartment. Following the S phase, the complex stage of prophase begins and these

become noticeably large.

The prophase of meiosis I spans 24 days and can be divided into the leptotene,

zygotene, pachytene stages, followed by the diplotene and diakenetic stages.

After this prolonged phase, comes the metaphase followed by the anaphase and

telophase. At the end of meiosis I, each primary spermatocyte divides into two

secondary spermatocytes which are haploid. The secondary spermatocytes then

proceed through meiosis II, at the end of which four haploid spermatids are formed.

Since meiosis II is not associated with DNA reduplication or recombination of genetic

material, it lasts a mere five hours, which is the reason why it is unusual to sight

secondary spermatocytes in a testicular biopsy.

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Spermiogenesis stage

In this stage, the round spermatid enters spermiogenesis, the third part of

spermatogenesis. During this process numerous nuclear and cytoplasmic changes

occur in the spermatids, eventually leading to the formation of the mature and motile

spermatozoa. Differentiation includes the following steps:

• Transit of the nucleus towards the periphery of the cell accompanied by

condensation of its content.

• Formation of the acrosome, which is in essence a modified lysosome. This attaches

to the nuclear surface at the region closest to the cell membrane.

• Formation of the flagellum, which includes the development of a microtubular core,

the axoneme, which arises from one of the centrioles of the round spermatid. Further

modifications of this structure yields the tail.

• Once this process is complete, the spermatid sheds a large part of its cytoplasm as

the residual body, which is phagocytosed by the Sertoli cell. The process of

spermiogenesis ends with release of the spermatozoa from the Sertoli cell. The

spermatozoa are still immotile when released and must mature further during storage

and transition in the epididymis.

Normal germ cell development and histology

The active element of the each testis consists of several hundred seminiferous tubules

up to 70 cm long. In these tubules spermatogenesis occurs. It is noted that all stages of

spermatogenetic differentiation can be identified simultaneously. Hence, it can be

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assured that spermatogenesis normally does not yield mature spermatids in every

tubular cross section in a testicular biopsy. A range of immature stages can be

normally appreciated and should not be considered an abnormality in maturation.

Therefore, several tubules have to examined before coming to a definitive conclusion.

The border of a tubule is a fine basement membrane flanked by strands of collagen

and elastin. The support system found external to the tubule is the interstitial tissue

constituted by mast cells, macrophages, Leydig cells, fibroblasts, vascular and

lymphatic channels. During puberty, Leydig cells, which bear the function of secreting

testosterone, mature within the interstitium.

The Leydig cells lie singly or are found in small clusters and are easily identified by

their granular eosinophilic cytoplasm. They may also normally contain small lipid

droplets, lipofuscin , or crystalloids of Reinke, which are rod like eosinophilic

inclusions in their cytoplasm.

The Sertoli cell is an intratubular elongated pyramidal cell with its base adhering to

the tubular basal lamina. Its function is to act as a supporter of spermatogenesis. The

Sertoli cells have poor cellular outlines, ultrastructurally this can be attributed to the

large number of lateral processes enveloping the spermatogenic cells. The mature

Sertoli cell contains a prominent centrally located nucleolus in its nucleus, useful for

quickly differentiating them from germ cells. In pathologic states, the nucleolus may

be less prominent, due to the Sertoli cell being in an immature or prepubertal state.

It is necessary to recognize the individual germ cell types, which proceed in an orderly

manner through spermatogenesis. The first stage is that of the spermatogonium, which

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is located near the basal lamina of the seminiferous tubules. It is a small cell (nearly

12 mm) with pale chromatin. As sexual maturity is attained, the spermatogonium

divides and either differentiates into type A spermatogonia, remains an

undifferentiated stem cell, or differentiates through mitotic cycles into a type B

spermatogonia. Type B spermatogonia give rise to the primary spermatocyte. The

primary spermatocyte undergoes its first meiotic division and goes through the four

prophase stages, leptotene, zygotene, pachytene, and diplotene, before reaching

meiotic metaphase. It takes nearly three weeks for the completion of the prophase

stages. Thus, numerous primary spermatocytes can be identified in an average cross

section of tubules in a typical biopsy. The primary spermatocyte is the largest germ

cell in the tubule, and the various stages are differentiated based on their degree of

chromosome coiling.

After meiosis, a smaller secondary spermatocyte is formed. This cell is occasionally

visible but typically seen fewer in number due to the short span of this stage. These

cells quickly undergo a second meiosis which gives rise to spermatids, which are only

7 to 8 mm in size. Spermatids are recognizable not just by their smaller size but also

by the fact that their chromatin is dark and condensed, and that they lie juxtaluminal

in their position within the tubule. Further differentiation results in the development of

a flagellum, progressive loss of cytoplasm, and elongation of their nucleus, which

gives rise to the mature spermatozoon, which is released into the lumen.

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Fig3: Histology of testis. Fig4: cross section of seminferous tubule.

http://www.lab.anhb.uwa.edu.au

PARACRINE CONTROL OF SPERMATOGENESIS

Hypothalamic-pituitary-testis circuit

The endocrine control of spermatogenesis is facilitated by the neuroendocrine activity

of the hypothalamic-pituitary-testicular axis. In man, there are three distinct phases,

spanning months to years, of postnatal activity along this axis that can be identified

before adulthood. The first phase is the neonatal-infantile phase that shows activity

similar to that seen in the adult phase along the hypothalamic-pituitary-testicular axis

but, remarkably, is not associated with spermatogenic activity. The second phase is

the prolonged juvenile/childhood phase when the hypothalamic-pituitary- testicular

axis is silent. In the final phase, the hypothalamic-pituitary-testicular axis activity is

http://www.lab.anhb.uwa.edu.au/

13

reinitiated and associated with pubertal initiation of spermatogenesis leading to

normal adult testicular functions. (7)

The crucial central, hypothalamic signal to the pituitary is the decapeptide

gonadotropin-releasing hormone (GnRH) that is secreted in a robust pulsatile manner

and acts through the transmembrane GnRH receptor. Subsequently, the two major

endocrine signals to the testis, originating from the pituitary gonadotropes that bear

GnRH receptors, are the distinct heterodimeric glycoprotein hormones FSH and LH.

Usually 70–90% of gonadotropes express both the common a and specific b subunits

of the gonadotropins and LH and FSH may be found within the same secretory

granules, which by a process of exocytosis deliver the gonadotropins to the peripheral

circulation. The gonadotropins are secreted in a pulsatile manner as a response to

GnRH. Comparatively, and in general, the pulsatile LH release is robust and similar to

that of GnRH while the pulsatile release of FSH is rather sluggish.

At the level of the testis, the two gonadotrophins, FSH and LH, mediate their actions

through specific transmembrane receptors, FSH-R and LH-R, respectively. Primarily,

FSH-R is expressed in the Sertoli cells in the seminiferous cords/ tubules while LH-R

expression is undergone in the interstitial Leydig cells. Both FSH (directly) and LH

(indirectly via testosterone-androgen receptor [AR]) exert their actions on

spermatogenesis mainly through the regulation of Sertoli cell factors.

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In reaction to the gonadotrophins, two major endocrine signals are produced from the

testis. These are the steroid hormone testosterone, produced by the Leydig cells in

response to LH signalling and secreted also in a pulsatile fashion, and the non-

steroidal hormone inhibin, produced by the Sertoli cells in response to FSH signaling

and secreted in a non-pulsatile manner.

Interestingly, substantiation by studies suggests an inhibitory role of testosterone (T)

on inhibin B secretion. In concert, these gonadal hormones are the chief feedback

signals that preserve the physiological function of the hypothalamic-pituitary axis. It

must be restated here that the usual role of gonadotrophins during puberty is chiefly to

establish the adult cohort of Sertoli, Leydig and stem germ cells and their tasks that

will ultimately lead to the normal spermatogenesis and sperm production. Hence,

hormone withdrawal during this phase will logically affect the normal scrotal descent

(where applicable) and development of the adult testis. Contrastingly, in the adult, the

effects of hormone withdrawal are primarily on the germ cells make-up via operative

impairments in the somatic cells, particularly the Sertoli cells.

The preceding intricacy of communications along the hypothalamic- pituitary-

testicular axis provides a reminder that the procedures of initiation and maintenance of

normal spermatogenesis are subject to destruction, due to toxic effects, not just

directly at the level of the testis but at a whole host of other, indirect sites along the

hypothalamic-pituitary-testicular axis.(8)

15

http://clinicalgate.com/tag/harrisons-principles-of-internal-medicine-

Fig5. Paracrine control of spermatogenesis

http://clinicalgate.com/tag/harrisons-principles-of-internal-medicine-

16

Paracrine control of gametogenesis

The seminiferous tubules of the mature mammalian testis are implicated in the

constant exocrine production of spermatozoa. Spermatogenesis, i.e. the maturation

and differentiation of diploid spermatogonia into fully developed haploid germ cells is

a complex process involving cell division of germ cell precursors by both mitosis and

meiosis, and their controlled dislodgement from the basal lamina into the tubule

lumen during their differentiation into mature spermatozoa. (9,10)

A group of spermatogonia are stimulated to initiate development at the same time and

these cells also proceed through spermatogenesis simultaneously. Throughout their

maturity the germ cells are closely associated with the neighbouring Sertoli cells,

which provide necessary nutritional and physical sustenance for gametogenesis. Each

Sertoli cell has to manage the task of concurrently meeting the nutritional and

metabolic needs of several germ cell generations in different stages of maturity. The

sequential relationships between the different maturing germ cell generations are

constant. Thus, specific maturational phases are always linked with each other leading

to the spermatogenic stages.(11) This controlled developmental direction of

consecutive germ cell generations with respect to each other is possibly necessary to

allow the Sertoli cell to simultaneously satisfy the nutritional and metabolic

requirements of various germ cell phases.

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Spermatogenesis is not synchronised throughout the seminiferous tubules. Discrete

frequently recurring variations in the stage of the spermatogenic cycle are observed

between different cross sections of the seminiferous tubules (the wave of the

seminiferous epithelium). (11) Such control is possibly the result of a rather

complicated paracrine interplay between the Sertoli cells and the germ cells. (9,12)

The initiation and preservation of testicular gametogenesis depends on FSH and

androgens. (9,13,14) Although there exists some contradicting data, (15) it is

generally thought that the Sertoli cells, but not the maturing germ cells, are directly

reactive to these hormones. Therefore, the control of spermatogenesis appears to be

almost completely reliant on normal Sertoli cell activity.

The aforementioned conclusion means that along with the well-recognized hormonal

interactions between the pituitary and the testis, supplementary paracrine control

mechanisms are essential for satisfying local needs in the seminiferous tubular

epithelium. Hence, it would appear that local communication between the Sertoli cells

and the maturing germ cells plays an significant role in the control of

spermatogenesis. Recent studies strongly recommend that this is the case.(9)

Sertoli cell cycle

Several discoveries suggest that the volume and ultrastructure of the Sertoli cell and

many aspects of its function vary according to the stage of the spermatogenic cycle.

Although the greater part of the S proteins secreted by the Sertoli cell are yet to be

identified, a few specific proteins like ASP, cyclic protein, transferrin and

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ceruloplasmin are known to be secreted cyclically during the spermatogenic cycle.

Recurring changes in Sertoli cell cytoskeletal structure, calmodulin concentrations,

FSH and androgen receptors, lipids and in the activity of several tubular enzymes and

in the secretion of MIS have also been encountered.

Although the functional importance of several of these cyclical processes remains

unidentified presently, in certain cases, it has been possible to exhibit that they mirror

the stage-specific needs of the maturing germ cells.

Also, the secretion of plasminogen activator and MIS is at its peak at the onset of

meiosis and the translocation of preleptotene spermatocytes through the tight Sertoli

cell junctions begins. There exists two kinds of plasminogen activators (PAs) in the

testis, i.e. tissue type and urokinase type.

PA secretion is under the control of FSH and retinoic acid (RA). It would seem that

the urokinase type PA is concerned with the opening of Sertoli cell junctions, thus

allowing the transduction of preleptotene spermatocytes to the adluminal

compartment. Preleptotene spermatocytes may locally stimulate the production of PA.

Since germ cells, but not Sertoli cells, possess most of cellular retinoic acid binding

protein. It is probable that the RA-induced transcription of urokinase type PA in

Sertoli cells is mediated indirectly via germ cells. The cyclicity in Sertoli cell

function may be regulated to a significant extent by the different germ cell phases, as

their depletion from the seminiferous epithelium adjusts Sertoli cell function, e.g. the

secretion of ASP and inhibin. (16,17)

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The careful destruction of preleptotene spermatocytes, but not of other maturing germ

cells, eliminates the stage - specific raise in plasminogen activator secretion. (18) Co-

culture studies indicate that different germ cell classes have divergent effects on the

function of Sertoli cells. (16,19,20)It would seem that Sertoli cell-germ cell

interactions involve means of cellular interaction necessitating cell-to-cell contact and

diffusible factors. (20)

Paracrine modulation within the interstitium; regulation of Leydig cell responsiveness,

steroidogenesis and blood flow

The intertubular compartment of the testis is made up of an extensive vascular system

meeting the high energy and oxygen requirements of spermatogenesis, and the

androgen producing Leydig cells as well as other cell types such as fibroblasts.

macrophages. mast cells and lymphocytes. The process of secretion and production of

androgens in itself, unlike spermatogenesis, is not the result of a sophisticated

interaction between different cell types. However. it would seem that the main need

for the paracrine modulation of Leydig cell function lies in the complete androgen

dependence of precise spermatogenic phases. (21,22)

Paracrine mechanisms may be required to guarantee adequately high local testosterone

concentrations near those tubular localities exhibiting androgen dependent phases in

spermatogenesis. Hence, testosterone has a dual role. i.e. it is a paracrine regulator of

spermatogenesis as well as being an endocrine hormone modulating other organs

through the systemic circulation. It is the first mentioned role of the Leydig cell that

makes paracrine control systems in the interstitium imperative.

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Leydig cell in spermatogenesis

The synchronization of peritubular Leydig cell function with the spermatogenic cycle

of the adjacent seminiferous tubule needs local chemical interaction between the

testicular compartments. Interruption of seminiferous tubule function has been shown

to bring about changes in Leydig cell morphology, LH receptors and steroidogenesis

.(23–26)Also the total volume of peritubular Leydig cells appears to change cyclically

according to the spermatogenic cycle. Leydig cell volume seems to be at its maximum

when the neighbouring seminiferous tubules exhibit the androgen dependent stages in

steroidogenesis.(27) These changes in the volume can be eliminated by

experimentally-induced spermatogenic distribution or unilateral cryptorchidism.

(27,28) Moreover in vitro studies with dissected tubules at specific stages demonstrate

that the androgen dependent stages have a considerable stimulatory effect on the

secretion of testosterone by purified but not crude Leydig cell preparations.(29,30)

The source of these tubular factors is possibly the Sertoli cell as both spent media

from Sertoli cell cultures and coculturing these cells with Leydig cells have been

observed to raise Leydig cell testosterone production and LH receptor numbers

.(20,31–33)

The chemical identity of these paracrine substances remains vague. An LHRH-like

factor presumably of Sertoli cell origin has been typified. (34–36) LHRH agonists

have been noted to have direct receptor-mediated effects on Leydig cell function. i.e.

short-term administration initiates testosterone secretion while long-term

administration (3 days or more) has inhibitory effects on steroidogenesis and

21

decreases LH receptor numbers. (22,34) The effects of this ligand on Leydig cell

function appear to be mediated by protein kinase C.(37) The LHRH -like factor may

act as a paracrine regulator of testicular microcirculation as well.(38–40). Hedger et

al, have provided supplementary evidence for the existence of a testicular LHRH-

peptidase most likely of Sertoli cell origin. (41)These studies propose a physiological

role for the LHRH-like factor in the testes of this species.

Significant difficulties have arisen, however, in the purification of this factor. Also,

efforts to find LHRH receptors in the testes as well as attempts to alter Leydig cell

function by blocking testicular LHRH receptors with antagonist have been

unsuccessful. The role of this substance as a modulator of Leydig cell function and

testicular microcirculation under normal physiological conditions remains unclear.

(42–44)

The seminiferous tubules synthesize local modulators of Leydig cell function other

than the LHRH-like factor which are primarily stimulatory in nature. (32,45,46) There

is also proof of there being an inhibitory factor modulating aromatization, which is

produced exactly at the androgen dependent stages of the spermatogenic cycle.(47)

Studies imply that Sertoli cells secrete numerous factors which modulate different

steps of the steroidogenic response of Leydig cells to LH stimulation. There is a

Sertoli cell-secreted protein which modulates Leydig cell adenylate cyclase, and one

or two supplementary factors which initiate testosterone and estrogen synthesis.

(33,48)The presence of luteinizing hormone receptor binding inhibitor, renin, POMC,

prodynorphin and CRF in the testis,(49–53) and the fact that Leydig cells bear specific

receptors to chemical effectors which regulate LH and steroidogenesis (e.g. prolactin,

22

glucocorticoids, insulin, benzodiazepine, epidermal growth factor and

catecholamines), (54) suggests that the modulatory communications regulating Leydig

cell function in the mammalian testis are extremely sophisticated. (2)

INDICATIONS FOR TESTICULAR BIOPSY:

Azoospermia or the complete absence of sperm in ejaculate, is generally the

reason for a testicular biopsy. This is the status of nearly 5% to 10% of men evaluated

for infertility and diagnosed when 2 consecutive sperm counts show 20 million sperm

per milliliter of seminal fluid or less. There may be several associated factors,

including defects in sperm motility, function, or chromosomal abnormality. It

represents the final outcome of a variety of testicular abnormalities, ranging from

normal spermatogenesis with seminal tract obstruction or absence of vas deferens

(obstructive azoospermia) to a range of problems with the spermatogenic process

itself (non-obstructive azoospermia).

Evaluation of men with azoospermia begins with a detailed history and physical

examination for gonad anomalies, followed by semen analysis, genetic studies, and a

hormonal profile, when possible. Unfortunately, these tests are sometimes

inconclusive. Hence, the importance of a testicular biopsy, which when properly

interpreted, becomes the major finding upon which a fertility specialist can base his

treatment plan for infertile men. Earlier, azoospermic men with levels of serum

follicle-stimulating hormone concentrations more than 2 to 3 times the normal were

23

termed as severe testicular failure not amenable to conventional therapy. A diagnostic

testicular biopsy was, in these cases, considered unnecessary. (55)

However, the onset of intra-cytoplasmic sperm injection using sperm harvested

through testicular micro-dissection, from the early 1990s, is now a viable treatment

option for many of these individuals. This technique is distinct from conventional in

vitro fertilization in that it needs only a single, viable spermatozoon per oocyte.

Therefore, many men who previously were not in vitro fertilization candidates are

now eligible for testicular biopsy and subsequent therapy. Still, the pivotal role of the

biopsy remains that of differentiating azoospermia due to obstructive aetiologies from

ablative testicular pathology. Biopsy is not warranted for cases in which the cause of

azoospermia can be elucidated on clinical grounds, such as Klinefelter syndrome or

prepubertal gonadotropin insufficiency.

A small yet distinct category of men with non-obstructive type of azoospermia shows

uniform maturation with normal testicular volume and normal levels of serum follicle-

stimulating hormone. These patients form a clearly distinct group with non-

obstructive azoospermia that have different treatment outcomes. They have a higher

incidence of chromosomal abnormalities and Y chromosome microdeletions as

against other men with non-obstructive azoospermia. Sperm retrieval may be difficult

and the chance of successful pregnancy is limited in these patients despite their having

normal follicle-stimulating hormone level and normal testicular volume.

24

HISTOPATHOLOGY OF TESTIS IN INFERTILITY

Germ cell abnormalities:

As per the recent recommendations published by the European Association of

Urology, for reporting a testicular biopsy, the different classes of germ cell

abnormalities are listed below. (56)

(1) absence of seminiferous tubules (seminiferous tubule hyalinization)

(2) presence of Sertoli cells only (Sertoli cell only syndrome)

(3) maturation arrest—incomplete spermatogenesis, not beyond the spermatocyte

stage

(4) hypospermatogenesis—all cell types up to spermatozoa are present, but there is a

distinct decline in the number of reproducing spermatogonia.

The etiology of most of these patterns cannot be determined from histology alone

because they each reflect a common manifestation of separate disorders that may be

more or less clear from clinical findings.(57)

Histological criteria for scoring testicular biopsies (according to the modified

Johnsen scoring)

(Score -10) Full spermatogenesis

(Score - 9 ) Slightly impaired spermatogenesis, many late spermatids, disorganized

epithelium

25

(Score - 8 ) Less than five spermatozoa per tubule, few late spermatids.

(Score -7 ) No spermatozoa, no late spermatids, many early spermatids

(Score - 6) No spermatozoa, no late spermatids, few early spermatids

(Score - 5) No spermatozoa or spermatids, many spermatocytes

(Score - 4) No spermatozoa or spermatids, few spermatocytes

(Score - 3) Spermatogonia only

(Score - 2) No germinal cells, Sertoli cells only

(Score - 1) No seminiferous epithelium(56)

Although, the Johnsen score is an easy and effective method to ensure uniformity in

the reporting of testicular biopsies, there have been noted to be several flaws. Firstly,

the system proposes to assess and grade the testis on the basis of loss of germ cells

from the most mature to the most basal. The problem arises when biopsies show a loss

of germ cell types at multiple levels of maturity. Secondly, the mean tubular score is

often not a true representation of the histologic picture. For instance, a complete

maturation arrest at the primary spermatocyte level and a mixed pattern of Sertoli cell

only and normal spermatogenesis (obstructive azoospermia) will both be scored 5.

However, it is evident that the management and outcome of the two conditions are

strikingly different. Hence, the use of the Johnsen score is dissuaded, although, it is

still widely in practice. (58)

26

Sertoli cell only syndrome

Sertoli cell only syndrome (also called germ cell aplasia) denotes a testicle from

which germ cells are absent, irrespective of stage, but the tubular architecture is intact

and the supporting cells are normally present. There is superficial resemblance of the

histopathological picture to the prepubertal testis. The aetiology can be varied. To

increase its clinical significance, the term Sertoli cell only syndrome is strictly applied

to the pattern where no germ cells are identified in any tubular cross section. In most

cases, the tunica propria and tubular basement membranes appear normal without any

hyalinisation. The tubules are also normal or show minimal decrease in diameter. The

interstitium contains varying population of Leydig cells(59).

The causes for this type of histology may be enumerated as follows:

Idiopathic( most common)

Karyotypic abnormalities such as Klinefelter syndrome

Treatment with hormones

Viral infections

Radiation

Toxin/chemical exposure

Congenital abnormalities

27

Fig6: Sertoli cell only

http://www.archivesofpathology.org(1)

Maturation arrest:

Alternatively termed germ cell arrest or spermatogenic arrest, this term should be

applied only when there is complete interruption of spermatogenesis uniformly in all

tubules. The arrest is most frequently encountered at the level of primary

spermatocytes, but can also sometimes be seen at earlier or later levels. There should

be spermatocyte maturation arrest uniformly in all the tubules visualised in each cross

section. This is rarely the case, hence a less strict definition is applied. This refers to

cases with focal spermatid maturation as ‗‗incomplete‘‘ maturation. These cases,

however, should ideally be classified as hypospermatogenesis with a heterogeneous

pattern.

28

Spermatogenic arrest can occur at spermatogonial level as seen in gonadotropin

insufficiency or due to germ cell damage by chemotherapy or radiotherapy.

Impairment of chromosome pairing during meiosis is the usual cause behind

maturation arrest at the spermatocyte level. Reversible arrest at the primary

spermatocyte level can be caused by various factors such as heat, infections, or

hormonal and nutritional factors, while irreversible arrest at the primary spermatocyte

or spermatid level is most often seen in chromosomal anomalies either in somatic cells

or in germ cells with subsequent impairment of meiosis.

Fig7: Maturation arrest.

http://www.archivesofpathology.org (1)

Hypospermatogenesis:

The presence of a mature spermatid in any tubule cross section indicates completion

of spermatogenesis and is a defining element of hypospermatogenesis, a disorder in

which complete maturation occurs, but the total number of germ cells is reduced. The

pattern can be homogenous across tubules, but more frequently there is variation

http://www.archivesofpathology.org/

29

among tubules including some showing extensive sclerosis or a Sertoli cell only

pattern. It is important to bear in mind that spermatogenesis does not appear complete

in every tubular profile in a biopsy even in the normal testis. Other clues to

hypospermatogenesis include an increased number of tubules with a smaller diameter,

and, due to the associated disruptions in the pattern of germ cell maturation, earlier

stages of germ cell development may be extruded into the lumen.

A wide number of factors cause the nonspecific change of irregular

hypospermatogenesis, including diabetes mellitus and the presence of a varicocele.

There are several predisposing conditions:

Hyperprolactinemia

Type 2 Diabetes Mellitus

Drugs such as cyclophosphamide

Hepatic disorders like cirrhosis

Substance abuse- alcoholism

Tescticular anomalies- varicocoele or undescended testis

Radiation

30

Fig 8: Hypospermatoenesis


Seminiferous tubule hyalinization:

Also termed ‗‗end-stage testis‘‘ or ‗‗tubular sclerosis,‘‘ these biopsies are identified by

extensive intratubular and peritubular hyalinization without the presence of germ

cells. Normal tubules are not evident. This pattern is distinguished from the mixed

patterns by its more substantial hyalinization, which includes all tubules within the

cross section. Sertoli cells are also frequently absent, although Leydig cells may be

seen in the interstitium. In testicular biopsies, these findings may be caused by

ischemia or remote infection, although in many cases an underlying cause cannot be

determined. The pattern may also be seen in adults with Klineflelter syndrome,

although these patients do not commonly require biopsy. The prognosis for patient

with infertility due to extensive tubular sclerosis is poor.


31

Fig. 9: Atrophy and peritubular fibrosis


Mixed patterns:

It is more frequent to see mixed patterns of testicular pathology than the

aforementioned examples. Commonly noted are Sertoli-cell–only syndrome or

hyalinized tubules in a background of seminiferous tubules with normal or decreased

maturation. As previously recorded, these findings should be categorized as

hypospermatogenesis with a percentage estimation given for each element. A

heterogeneous pattern impedes the diagnosis of maturation arrest, which is uniform

across all tubules. A mixed pattern of hyalinized and Sertoli-cell–only tubules with

some tubules consisting of immature or prepubertal Sertoli cells has been suggested as


32

a characteristic of Klinefelter syndrome, but similar patterns can be also encountered

in germ cell aplasia or varicocele, so the diagnosis of Klinefelter syndrome should not

be made solely on the basis of testicular biopsy. A decrease in testicular function

occurs normally along with ageing, matched by involutional changes in the testicular

parenchyma, including hypospermatogenesis, peritubular fibrosis, and hyalinization of

tubules commonly resulting in a pattern resembling that of a mixed primary testicular

pathology. Although a few sclerosed tubules may still be compatible with a normal

aging testis, larger or contiguous areas of sclerosis are evidently pathologic. Abnormal

sperm maturation, sloughing of germ cells in the tubular lumen, degeneration of germ

cell elements, and Sertoli cell lipid accumulation and cytoplasmic vacuolization are

frequent.(1)

Fig. 10: Mixed pattern showing Sertoli cell only pattern in one tubule with normal

spermatogenesis in the background



33

Fig. 11 ALGORITHM FOR EVALUATION OF TESTICULAR BIOPSY IN

INFERTILITY

34

Y CHROMOSOME MICRODELETION

Fig.12: Schematic representation of Y chromosome

Fig. 13: Representation of the cytological bands of the Y chromosome.

35

Genetic aetiologies have a distinct role in male infertility. As much as 12% of men

with non-obstructive azoospermia have karyotypic anomalies(60), while Y

chromosome microdeletions are identified in 6±18% of men with non-obstructive

azoo- or oligozoospermia.(61–65) A prominent part for the Y chromosome in

spermatogenesis was ascertained nearly 25 years ago when Tiepolo and Zuffardi (66)

recognised, on karyotyping, significant terminal deletions on the long arm of the Y

chromosome (Yq) in six men with azoospermia, suggestive of factors (azoospermia

factors) required for spermatogenesis to be found in the euchromatic part of Yq.

Successive cytogenetic(67,68)and molecular(69–71) studies effected in the explication

of multiple terminal and interstitial deletions of the Y chromosome related to

abnormal spermatogenesis.

Structure of the male-specific region of the y chromosome (MSY)

The complete physical map and sequence of MSY have been available since

2003.(72) This information was derived by sequencing and mapping 220 BAC clones

which had portions of the MSY from one man. The use of only one individual was

necessary because, due to the presence of repetitive sequences with only minute

differences characterizing the individual copies of each sequence (sequence family

variants, SFV), inter-individual allelic variation or polymorphisms would have

prevented the precise mapping of SFV required to allocate the BAC clones. Three

classes of sequences were foundin MSY: X-transposed (with 99% identity to the X

chromosome), X-degenerate (single-copy genes or pseudogene homologues of X-

36

linked genes) and ampliconic. Ampliconic sequences are identified by sequence pairs

showing nearly complete (>99.9%) identity, arranged in massive palindromes.

According to present knowledge, the reference MSY contains 156 transcription units

including 78 protein-coding genes encoding 27 proteins. Ampliconic sequences are

constituted by 60 coding genes and 74 non-coding transcription units predominantly

grouped in families and expressed mainly or only in the testis. Ampliconic sequences

recombination takes place through gene conversion, that is, the non-reciprocal transfer

of information about sequences occurring between duplicated sequences within the

chromosome, a process which maintains the >99.9% identity between repeated

sequences organized in pairs in inverted orientation within palindromes.

Apart from maintaining the gene content, this particular sequence organization gives

the structural edifice for deletions and rearrangements. It is widely acknowledged that

complete AZF deletions occur invariably through Non-Allelic Homologous

Recombination (NAHR) which takes place between highly homologous repeated

sequences with the same orientation leading to loss of the genetic material between

them.

Considering the architecture of the MSY, many varied deletions are hypothetically

possible(73–75) and those which are clinically important for male infertility, based on

present knowledge, are briefly described below.

37

Mechanism and type of deletions

Three discrete AZFa, AZFb and AZFc regions were originally typified by careful

mapping of the MSY of a large number of men with microdeletions when the

sequence of the Y chromosome was not entirely known.(63) Consequently, due to

molecular description of the deletions, a new representation of deletions, in which the

AZFb and AZFc regions are found to overlap, has been suggested. In addition, the

AZFb and AZFbc deletions have been proposed to be the consequence of at least three

different deletions patterns.(76) While the new nomenclature is more appropriate in

biological terms, from the practical, clinical standpoint either nomenclature can be

adopted for the complete AZFb (P5/proximal P1) and AZFc (b2/b4) deletions.

Contrastingly, the difference between the two AZFbc subcategories (P5/distal P1 and

P4/distal P1) does have clinical relevance. We additionally provide information on the

genomic restriction of the sY-loci used for the AZF analyses which should be used to

depict deletions following the HGVS nomenclature as part of a standard practice.

The AZFa region is about 1100 kb long and contains the single-copy genes USP9Y

(former DFFRY) and DDX3Y (former DBY). Present data acquired concurrently by

various groups identify the source of complete AZFa deletions in the homologous

recombination between identical sequence blocks within the retroviral sequences in

the same orientation HERVyq1 andHERVyq2. (76-78)Within these retroviruses,

recombination can happen in either one of two identical sequence blocks (ID1 and

ID2), producing two major pattern of deletions slightly different in their precise

breakpoints.(78–80) In any case, the total deletion of the AZFa region eliminates

about 792 kb including both USP9Y and DDX3Y genes, the only two genes in the

38

AZFa region.The type and mechanism of deletions of the AZFb and AZFc region

have been clarified by Kuroda-Kawaguchi et al. (73)

Both regions together are constituted of 24 genes, most of which are found in repeated

copies for a total of 46 copies. The whole deletion of AZFb removes 6.2 Mb

(including 32 copies of genes and transcription units) and is the result of homologous

recombination between the palindromes P5/proximal P1. (81)The AZFc region

comprises 12 genes and transcription units, each presents in a variable number of

copies making a total of 32 copies.(75) The classical total deletion of AZFc, the most

common pattern among men with deletions of the Y chromosome, removes 3.5 Mb,

arises from the homologous recombination between amplicons b2 and b4 in

palindromes P3 and P1, respectively, and removes 21 copies of genes and

transcription units.(73)

Deletions of both AZFb and AZFc in concert occur by two major means involving

homologous recombination between P5/distal P1 (7.7 Mb and 42 copies removed) or

between P4/distal P1 (7.0 Mb, 38 copies removed).(81) Therefore, according to the

current knowledge, the following repeated microdeletions of the Y chromosome are

clinically relevantand are found in men with severe oligo- or azoospermia:

• AZFa,

• AZFb (P5/proximal P1),

• AZFb&c (P5/distal P1 or P4/distal P1),

• AZFc (b2/b4).

39

The most common deletion type is the AZFc region deletion (~80%) followed by

AZFa (0.5–4%), AZFb (1–5%) and AZFb&c (1–3%) deletion. Deletions which are

identified as AZFa,b&c are most probably related to abnormal karyotype such as

46,XX male or iso(Y).(82)

Gr/gr deletion

The AZFc region is particularly susceptible to NAHR events which are known to

cause partial deletions and duplications leading to gene dosage variations.(73,74,83)

The most important clinical partial deletion is the ‗gr/gr‘ deletion, named after the

fluorescent probes (‗green‘ and ‗red‘), first described. (75) Although it removes half

of the AZFc gene content (genes with exclusive or predominant expression in the

germ cells), its clinical significance is still a matter of debate, because carriers tend to

manifest varying spermatogenic phenotypes which can range from azoo- to

normozoospermia. Evidently the effect of this deletion mostly depends on the ethnic

and geographic origin of the populations studied. In fact, the frequency and

phenotypic effect may vary among different ethnic groups, based on the Y

chromosome background. For example, in Japan and certain areas of China, where

the most common specific Y haplogroups are D2b, Q3 and Q1, the gr/gr deletion

does ot significantly affect spermatogenesis.(84,85)

Controversies are also related to selection biases (lack of ethnic/ geographic matching

of cases and controls; inappropriate selection of infertile and control men) and

methodological issues (lack of confirmation of gene loss). Many attempts have been

40

made to explain the molecular basis for the highly varied phenotypic arrangement of

this deletion type. It has already been illustrated that the loss of DAZ1/DAZ2 and

CDY1 is common (or even distinct) in carriers with poor sperm production

(86,87)while it was suggested that the restoration of normal AZFc gene dosage in case

of gr/gr deletion followed by b2/b4 duplication may illustrate the poor effect on sperm

count.(75,88) With respect to this, a large multicentric study was done, which was

based on a combined method (gene dosage, definition of the lost DAZ and CDY1

genes, Y hgr definition). The study was carried out among Caucasians.(89) Despite

the detailed description of subtypes of gr/gr deletions on the basis of type of absent

gene copies and the identification of secondary rearrangements (deletion followed by

b2/b4 duplication) along with the definition of Y haplo-groups, defining a specific

pattern which could be associated with either a ‗neutral‘ or a ‗pathogenic‘ effect was

unsuccessful. On the other hand, studies among Asian populations appear to support

the hypothesis about a deletion subtype-dependent phenotypic effect and about the

significance of the Y background in which the deletion arises. (85,90)Along with the

classic case/control studies aiming at defining whether the gr/gr deletion presents a

risk for spermatogenic disturbances, the examination of successive patients through

cross-sectional cohort analysis suggests that the gr/gr deletion has an effect even

within the normal range of sperm count. It was detected that normozoospermic

carriers have a significantly lower sperm count, as against men with intact Y

chromosome.(91) Also, Yang et al. (92) stated that, among the Chinese population,

the deletion frequency is dramatically reduced in subgroups with sperm concentrations

>50 9 106/mL.

41

The screening for gr/gr deletion is based upon a PCR plus/minus technique of two

markers (sY1291 and sY1191)(75) and the diagnosis depends on the absence of

marker sY1291 and presence of sY1191. It is meaningful to identify that there is a 5%

possibility of false deletion rate being detected in the multicentric study,(89)

emphasizing the significance of optimizing PCR conditions and of using

supplementary confirmatory measures like simplex PCR and eventually gene dosage

analysis (Giachini et al., 2005; Choi et al., 2012).(88,90) The definition of the Y

haplogroup is suggested in Asian cohorts to prevent the inclusion of constitutive

deletions which are unlikely to affect spermatogenesis.

Isolated AZF gene-specific deletions

Even though some authors have found a high frequency of single AZF gene

deletions,(93,94) these data contradict the general experience accumulated in >2000

patients tested elsewhere. (95–99)Gene-specific deletions are very rare, and all the

five confirmed deletions (with the definition of the breakpoints) removed completely

or partially the USP9Y gene belonging to the AZFa region.(100)None of the deletions

occurred due to NAHR and thus are likely to bedistinct, supporting the fact that these

occurrences are rare. The associated phenotype of semen/testis is largely varied

among USP9Y deletion carriers (from azoospermia caused by hypospermatogenesis to

normozoospermia) implying that this gene acts as a fine tuner rather than as an

essential factor for spermatogenesis.

Based on the absence of other than USP9Y gene deletions in the literature, screening

for isolated gene-specific deletions is not advised in the routine diagnostic setting.

42

Although some of the commercially available kits have gene-specific markers, a lot of

discretion has to be taken both in the validation and interpretation of suspected single-

gene deletions.

Correlation between the genotype and phenotype of complete AZF deletions:

AZF deletions are specific for spermatogenic failure as no deletions have been

reported in a large number of normozoospermic men.(99,101) Fertility being

sometimes compatible with these deletions, emphasizes that natural fertilization may

occur even with low sperm counts depending on the status of female partner‘s fertility.

Therefore, it is more suitable to deem Y chromosome deletions as a cause for

oligo/azoospermia instead of being considered a cause of ‗infertility‘.

Deletions of the complete AZFa region consistently result in Sertoli cell only

syndrome (SCOS) and azoospermia. (62, 79, 101–103)The diagnosis of a total

deletion of the AZFa region indicates how difficult it is to retrieve testicular

spermatozoa for intracytoplasmic sperm injection (ICSI).

Complete deletions of AZFb and AZFbc (P5/proximal P1, P5/distal P1, P4/distal P1)

are typified by a corresponding histology of SCOS or spermatogenetic arrest resulting

in azoospermia. Many reports have depicted that similar to the complete deletions of

the AZFa region, no spermatozoa are found whenattempts are made for testicular

sperm extraction (TESE) in these patients. (102,103,105)However, in three cases,

reports of spermatid arrest and even crypto/oligozoospermia have been the result in

association with complete AZFb or AZbc deletions.(106,107)There is no definite

biological explanation for the unusual phenotypes. It has been suggested that both the

43

background effect of Y chromosome and the differences in the finite extent of the

deletions may be causative. Indeed, a smaller deletion, that is, a proximal breakpoint

at P4 may be related to the retention of AZFb gene copies such as XKRY, CDY2 and

HSFY. Hence, it has been proposed that the correlating phenotype is more critical in

case of complete removal of the AZFb region. With very few exceptions stated in

literature, the diagnosis of complete deletions of AZFb or AZFbc (P5/proximal P1,

P5/distal P1, P4/distal P1) means that the chances for testicular sperm retrieval is nil

even with micro-TESE.(64)

Varied clinical and histological phenoytpes are identified with deletions of the AZFc

region (b2/b4).(62,108,109) Generally, AZFc deletions can be found in men with even

severe degrees of oligospermia as they are found to be compatible with residual

spermatogenesis. It has also been noticed to be rarely transmitted naturally to the male

offspring. (110)In men with azoospermia and AZFc deletion there is approximately

50% chance of retrieving spermatozoa from TESE and conception can be attained by

ICSI.(99,109,111–120) The rates of success with TESE are hugely dependent on the

technique used and can be as low as 9% (120) or as high as 70–80% following micro-

TESE. (103)

44

PATIENTS AND METHODS:

There were 70 males who underwent testicular biopsies for evaluation of infertility

during the period spanning Jan 2010 to Dec 2014.

The following clinical and laboratory features were recorded:

Age;

Testicular volume*;

Semen analysis;

Serum levels of FSH, LH, testosterone and prolactin.

*Testicular volume was assessed clinically. Ultrasosnographic estimation was not

done. testicular volumes were categorised as follows:

normal (15-25 ml);

sub-normal (12 -15);

critically low (less than 12 ml).

All patients had undergone open biopsies. Under local anaesthesia, a scrotal incision

was made and the tunica albuginea was opened. Gentle pressure was applied so as to

allow tissue to protrude out from the incision. This tissue was excised and

immediately immersed in Bouin‘s fixative for 12 hours. The tissue was then immersed

in toluidine buffer solution following which it was transferred to graded alcohol for

routine processing and later embedding in paraffin.

45

Slides were prepared from 5-micron sections and stained using hematoxylin and

eosin.The following special stains were used as required: Periodic acid Schiff (PAS)

stain to highlight thickening of the basement membrane and Ziehl-Neelsen stain to

identify acid fast bacilli.

Microscopy: The number of tubules was counted using the 10X objective. Sections

containing 20 or more seminiferous tubules were considered adequate for histological

examination. The morphology of the tubules and interstitium were studied using the

high power objective (40X).

The following parameters were studied histologically:

number of tubules;

tubule diameter*;

tubular basement membrane thickness*;

presence of tubular atrophy and /or hyalinisation;

prominence of Sertoli cells;

degree of maturation of germ cells;

interstitial cellularity and vascularity;

number of Leydig cells;

presence or absence of fibrosis.

46

*Morphometry was used to estimate tubule diameter and basement membrane

thickness, using Cellsens image analysis software. Each slide was assessed at 25X

magnification and five such fields were arbitrarily selected, for calculating the

diameter. A mean was calculated for each case. The tubule diameter was considered

normal if ≥150 microns. (121)

The basement membrane thickness was considered to be normal if ≤0.40

microns(normal range 0.3 to 0.4microns).(122)

Based on the distribution of Leydig cells in the interstitium different patterns were

observed, normal Leydig cells, Leydig cell clusters(<10 cells), Leydig cell hyperplasia

(if more than 10-20cells per cluster) (121)

Conventional cytogenetic analysis (peripheral blood karyotyping) was done using

standard protocols. (123)Briefly, peripheral blood was collected using sterile

precautions in a sodium heparin vacutainer and cultured for 72 hours after

phytohaemagglutinin-stimulation, following which the chromosomes were harvested.

The harvested chromosomes were banded (stained) with Leishman stain following

treatment with trypsin (trypsin G-banding). At least 20 G-banded metaphases were

analysed for each patient.

Fluorescence in situ hybridization (FISH) analysis was performed when required to

better describe an abnormality. The probes used were as follows: a whole Y

chromosome paint, probes to the centromeres of chromosomes X and Y, and locus

specific probes for the SRY gene and the terminal ends of both arms of chromosome 1

(bands 1p36 and 1q44 ). The probes were obtained from Abbot Vysis ( Illinois,

47

U.S.A.. For FISH analysis, 100 interphase cells and 20 metaphases were

studied. Standard protocols were used for both karyotyping and FISH. (124,125)

Conventional cytogenetic analysis and FISH analysis were done using Zeiss Axioskop

and AxioImager microscopes at 100X magnification and automated karyotyping

system (Ikaros and Isis, MetaSystems GmbH, Altlussheim, Germany). Results

were reported in accordance with the International System for Human Cytogenetic

Nomenclature (ISCN). (126)

Y chromosome microdeletion analysis was done on peripheral blood collected in

EDTA using a multiplex polymerase chain reaction (PCR) in accordance with the

guidelines of the European Academy of Andrology and the European Molecular

Genetics Quality Network using the following sequence tagged sites (STS) loci:

AZFa(sY84,sY86), AZFb(sY127, sY134), AZFc (sY254, sY255) with ZFY and SRY

as controls.

48

RESULTS:

There were 70 patients who underwent testicular biopsies for investigation of

infertility. Only one testis was biopsied in all but two patients. Two patients had

bilateral biopsies done. All other patients had only biopsy of one side.

The clinical and laboratory features are described below.

1. Age:

The age distribution of our patients is shown in Table 1. The patients ranged from 23 -

47 years of age, with a mean age of 33 years. The majority of patients (44%)

belonged to the age group of 31to 35 years.

Table 1: Age distribution

Age (years) No. of patients (%)

21-25 3 (4)

26-30 16 (23)

31-35 31 (44)

36-40 14 (20)

41-45 5 (7)

45-50 1 (2)

49

2. Testicular volume:(Table 2)

Patients were categorised into three groups according to the testicular volume

which was assessed clinically. These categories were normal (15-25 ml),

subnormal (12 -15) and critically low (less than 12 ml).

Testicular volumes were available for 44 of our patients and ranged from 1.5 ml to

20 ml (mean 11 ml). Only 36% of our patients had normal testicular volumes. The

majority (55%) had critically low volumes.

Table 2: Testicular volume.

Categories Number(%)

Critically low(1-12ml) 24(55)

Sub-normal (12-15ml) 4(9)

Normal (15-25ml) 16(36)

3. Semen analysis: (Table 3)

There were 66 patients (94%) with azoospermia. The remaining four had

oligospermia, One of these patients had a sperm count of 10 million/ml, with 50-

60% motile sperms and 44% normal sperms.In the remaining three patients with

oligospermia, the exact counts were not available because the semen analysis was

done elsewhere. One patient with oligospermia was suspected to have tuberculous

orchitis.

50

Table 3: Results of semen analysis

Semen analysis Number of patients (%)

azoospermia 66(94)

oligospermia 4(6)

4. Hormonal profile:(Table 4)

Serum levels of FSH were done in all 56 patients who were evaluated in our

centre. However, the 14 patients who were initially evaluated elsewhere and sent

here for further management did not have records for the same. Serum levels of the

other three hormones expected to be evaluated in this group of patients were

available for variable numbers of patients, as shown in the table

The mean FSH value was 15.5 (range 2.42-56.4). The majority of the

patients(59%) had high FSH values(>11mIU/ml).

Table 4: Hormonal profile

Hormone No. of patients Range Mean

FSH

(normal 07-11.1 mIU / ml)

70 2.42-56.4 15.5

LH

(normal 0.8-7.6 IU/ml)

1 5.5 -

Testosterone

(normal 33.8-106%)

5 201-691 415.6

Prolactin

(normal 2.5-17 ng/ml)

6 4.6-12.5 7.9

51

Table 5: Distribution of patients according to FSH levels.

Normal (mean 6.4) Elevated FSH levels (mean 21.8)

FSH level

(mIU/ml) 0.7-11 11-20 20-30 >30

Number

of patients 23 17 13 3 (35.44.5,56.4)

5. Histopathological examination:

5.a) Tubule number:

All but one of our biopsies had an adequate number of seminiferous tubules (range

20 -500, mean 78). In one patient whose biopsy was considered inadequate, only

four tubules were seen.

Table 6: Classification based on number of seminiferous tubules in the biopsy

Number of tubules Number of cases(%)

Adequate

20-100 52(74)

100-200 15(22)

>200 1(2)

Inadequate

<20 1(2)

52

5.b) Morphometric assessment of seminiferous tubules:

5.b.i ) Tubule diameter:(Table 7)

The tubule diameter was assessed by morphometry and ranged from 44 – 487

microns (mean 216 microns).

5.b.ii)Basement membrane: (Table 7)

Basement membrane thickness was also evaluated by morphometry and ranged

from 0.14 to 0.69 microns.

The basement membrane was thickened (more than 0.40 microns) in 26

cases(38%) and normal in 43(62%).

Table 7: Tubule diameter and basement membrane thickness

Parameter Number (%) Mean

Tubule diameter(microns) 216

Normal ( ≥ 150 µ ) 16(23) 249

Reduced ( ≤ 150 µ ) 53(77) 110

Basement membrane(microns) 0.45

Normal (0.3– 0.4 microns) 43(62) 0.3

Thickened (≥ 0.4 microns) 26(38) 0.49

53

5.c) Interstitium(Table 8)

Interstitial cellularity and vascularity were assessed.

Interstitial cellularity and vascularity were assessed and categorised as shown in Table

8. The interstitium was normal in the majority of patients (76 %).The other features

were seen in <5-10% of our patients as shown in the Table 8. Three biopsies showed

epithelioid cell granulomata associated with lymphohistiocytic aggregates; acid fast

bacilli were not seen. One of these patients was suspected to have tuberculosis prior to

the biopsy. In the other two, the granulomata were an incidental finding.. All three

cases with Leydig cell hyperplasia and one with Leydig cell clusters, were associated

with an SCO pattern, as described in section 5.f.iv. Fibrosis and vascular sclerosis

were noted in <5% of biopsies.

Table 8: Features of the interstitium

^ includes one patient with SCO, * all with SCO.

Features of interstitium Number of patients(%)

Cellularity

Normal with lymphocytes 52 (76)

Leydig cell clusters (<10 cells) 5^ (8)

Leydig cell hyperplasia (>10 cells) 3* (4)

Granulomata 3 (4)

Other

Fibrosis 3(4)

Scant or absent with no fibrosis 3(4)

Vascularity

Normal 67(97)

Thickened 2(3)

54

5.f) Histology of seminiferous tubules(Table 9)

The histological findings were categorized based on the characteristics of the

germinal epithelium as follows:

Table 9. Histology of seminiferous tubules

Diagnosis Number(%)

Normal spermatogenesis 8 (12)

Hypospermatogenesis 11 (16)

Maturation arrest 28 (40)

Sertoli cell only 20 (29)

Atrophy/Fibrosis 2 (3)

5.f.i)Normal spermatogenesis:

This pattern was seen in 8 biopsies(12%), all of whom had preliminary

investigation elsewhere and were referred her for further management. The

patients ranged in age from 26 – 38 years. FSH levels were available in only two

patients, both of which were normal. These biopsies revealed all stages of germ

cell maturation, upto mature spermatozoa, as described below.

a) Spermatogonia, the undifferentiated germ cells, located along the basal lining of

the seminiferous tubule. Spermatogonia were of two histologic types-

spermatogonia A, with an oval nucleus bearing condensed chromatin and

peripherally placed nucleoli.

55

spermatogonia B, with dispersed chromatin revealing centrally placed

nucleoli.

b) Primary spermatocytes, the next stage of cells displaying abundant

eosinophilic cytoplasm, large nuclei and coarse chromatin.

c) Secondary spermatocytes are smaller than primary spermatoytes, with a

smudged nucleus, are rarely encountered in a tubule as they undergo

rapid meiosis, forming the spermatids.

d) Spermatids: these are extremely small cells with a hyperchromatic

central nucleai and are found adluminally.

e) Mature spermatozoa- In the centre of the lumina are found the

spermatozoa with their small and pointed nuclei and their elongate tail.

The tubules showed varying degree of maturation in different tubules within the

same field. This was in keeping with normal spermatogenesis. The presence of

normal spermatogenesis implies that azoopsermia was due to obstruction to the

passage of sperms. The details of these patients are available in Appendix.

5.f.ii) Hypospermatogenesis :

This pattern was seen 11 biopsies (16%). The picture was similar to normal

spermatogenesis. However, we found that

The number of cross sections showing maturation upto mature spermatozoa

was decreased;

The number of mature spermatozoa found in each tubule was also markedly

decreased to only one or two spermatozoa per tubule.

56

In 6/11 cases (9%), maturation extended only upto the spermatid stage, with

only a few spermatids seen.

These findings were in accordance with the modified Johnsen scoring system

which states that a biopsy to be categorised thus, there must be less than 5

mature spermatozoa per tubule.

Profile of patients with hypospermatogenesis Table10.

UID AGE TEST

VOL FSH KARYOTYPE Y CHR

10 33 13.5 6.2 . .

12 29 . . . .

13 31 12 7.4 . .

15 43 17 22.8 . .

19 38 . . . .

22 31 15 6.83 . .

26 29 10 16.6 . .

32 36 20 6.46 . .

37 29 15 8.46 46, XY Negative

49 41 . 14.2 46, XY Negative

63 35 12 15.8 46, XY Negative

57

5.f.iii) Maturation arrest: (Table 11)

These biopsies showed complete interruption of spermatogenesis in all tubules

visualised in each cross section, uniformly. The maturation arrest was most

frequently encountered at the level of primary spermatocytes (57%). In the

remaining 43 %, maturation arrest was encountered at the level of secondary

spermatocytes and spermatogonia.

Table 11. Stages of maturation arrest

Maturation arrest N=28(%)

Primary spermatocyte 16(57)

Secondary spermatocyte 3(11)

Spermatogonia 9(32)

Table 12: Profile of patients with maturation arrest.

UID AGE TEST

VOL FSH

LEVEL OF

ARREST KARYOTYPE

Y CHR

DELN

4 29 . . Primary

spermatocyte . .

5 29 20 28.6 Primary

spermatocyte . .

9 34 13 13.7 Primary

spermatocyte 46,XY[40] Negative

11 32 12 15.9 Primary

spermatocyte . .

14 35 15 2.58 Primary

spermatocyte 46,XY[20] AZFb

17 33 1.5 2.38 Primary


24 32 15 4.79 Primary

spermatocyte . .

30 37 15 27.5 Primary

spermatocyte . .

58

33 25 20 4.09 Primary

spermatocyte . .

36 39 . 3.02 Primary

spermatocyte . .

39 27 10 20.8 Primary

spermatocyte . .

41 30 15 21 Primary


42 34 . 21.6 Primary

spermatocyte . .

47 26 9 25.1 Primary

spermatocyte . .

57 33 10 16 Primary

spermatocyte . .

64 26 . . Primary

spermatocyte . .

35 37 10 14.7 Secondary


62 35 . . Secondary

spermatocyte . .

67 35 . 4.85 Secondary


16 31 20 2.43 Spermatogonia . .

20 36 7.5 4.86 Spermatogonia 46,XY[20] AZFb

21 28 . . Spermatogonia . .

27 36 10 13 Spermatogonia . .

46 36 10 25.1 Spermatogonia . Negative



60 32 13 4.65 Spermatogonia 46,XY[20] Negative

68 31 9 18.3 spermatogonia 46,XY[20] Negative

59

5.f.iv) Sertoli cell only :

This pattern was seen in 20 biopsies (29%) and was characterized by the complete

absence of intratubular germ cells. The Sertoli cells appeared as ‗palm trees in a

breeze‘ or ―wind-swept tree tops‖, and were aligned perpendicular to the basement

membrane. The cells were oval in shape and the nuclei were irregular or folded

with prominent nucleoli. In most cases, the tunica propria and tubular basement

membranes appeared normal , although in a few cases hyalinization was seen The

interstitium contained varying numbers of Leydig cells.. One case showed Leydig

cell clusters and three showed Leydig cell hyperplasia. The others did not show

any prominence of these cells.

Table 13: Profile of patients with Sertoli cell only.

UID AGE TEST VOL FSH KARYOTYPE Y CHR

1 27 . . 1 .

2 29 20 13.5 46,XY[20] Negative

3 33 . 44.5 . .

6 32 12.5 14.5 46,XY[20] Negative

7 25 10 9.96 46,XY[20] Negative

8 41 10 10.8 . .

18 31 . 35 . .

23 35 12 5.13 . .

31 37 12 23.6 . .

34 32 15 18.6 46,XY[20] Negative

38 37 8 15.2 46,XY[45] Negative

43 33 10 8.97 . .

45 47 10 27.3 46,XY[20] Negative

53 31 . 26.6 46,XY[20] Negative

55 30 . . . .

58 31 . 26.6 46,XY[20] Negative

59 43 15 56.4 . .

61 23 8 8.79 45,X,add1q44[25]

AZFa,b,

c

65 39 . 15.5 . .

69 31 10 26.6 46,XY[20] Negative

60

5.f.v) Peritubular fibrosis and atrophy:

This pattern was seen in 2(3%) cases, one which showed complete atrophy of

tubules with no intatubular germ cells and associated fibrosis of the interstitium.

The other case showed a few scattered Leydig cells in the interstitium.

Table 14: Profile of patients with peritubular fibrosis

UID AGE TEST VOL FSH KARYOTYPE Y CHR

29 37 . 10.2 . .

44 29 9 15.3 . .

61

Fig.14 (a&b): a.Granulomatous inflammation, H&E, 10X. b. Another region of the

same testis showing normal spermatogenesis, H&E, 40X..

62

Fig. 15: Normal distribution of Leydig cells(black arrow) and lymphocyte(red

arrowheads), 20X..

Fig. 16: Leydig cell clusters

63

Fig. 17: Leydig cell hyperplasia(black arrow) with peritubular fibrosis(red arrow),

H&E,20X.

Fig. 18: Leydig cell hyperplasia, H&E, 20X

64

Fig. 19: Normal spermatogenesis, 20X.

Fig. 20: Normal spermatogenesis, 40X( black arrow-spermatogonia, red arrow-

primary spermatocyte, blue arrow- spermatid)

65

Fig. 21: Hypospermatogenesis showing occasional mature spermatozoa,

H&E,40X

Fig. 22: Maturation arrest at the level of spermatogonia( black arrow-

spermatogonia A, red arrow- spermatogonia B), H&E,40X

66

Fig.23: Maturation arrest at the level of primary spermatocyte, H&E,40X

Fig. 24: Maturation arrest at the level of secondary spermatocyte, H&E,25X

67

Fig.25: Sertoli cells only, H&E, 40X

Fig. 26: Atrophy of seminferous tubules, H&E10X.

68

Fig. 27: Peritubular fibrosis, H&E, 25X

Fig.29: Karyotype of 45,X male, with SRY gene insertion on chromosome 1.

69

Fig. 30: FISH probe showing absence of signal for Y chromosome.

Fig. 31: FISH probe showing insertion of SRY gene at long arm of chromosome 1.

70

Fig. 32: Y chromosome microdeletion study showing deletion of the AZF b

Fig. 33: Y chromosome microdeletion in AZF a, b & c regions, in 45, X male.

SRY/ZFY

AZF b

AZF c

AZF a

Mal

e co

ntr

ol

Pt w

ith

AZ

F a

, b

& c

del

etio

n

Pt w

ith

AZ

F a

, b

& c

del

etio

n

Mal

e co

ntr

ol

Fem

ale

con

tro

l

Bla

nk

Bla

nk

Fem

ale

con

tro

l

71

Correlation between histology and other parameters

6. Age and histology: There was no significant correlation between the two

parameters (p=0.9).

7. Testicular volume and histology: The mean testicular volumes invarious

histological groups were compared to one another.There was no correlation between

testicular volume and histology.(p= 0.2)

Table 15: Comparison of testicular volumes across the various histological profiles

Diagnosis Testicular volume(ml)

Sertoli cell only 11.5

Maturation arrest 11.4

Hypospermatogenesis 13.5

FSH and histology: It was noted that in both the normal and high categories of FSH,

the highest percentage of cases was maturation arrest. This can be explained by the

fact that this category was the major histological subtype in our sudy. However, t was

interesting to note that in the group with high FSH values, Sertoli cell category was

almost as significant. There were 14 cases with maturation arrest and 13cases with

Sertoli cell only in this category. In the group with the extremely high values for FSH,

there was only one histological subtype and that was Sertoli cell only, as expected.

72

Table 16: FSH variation across various histological profiles.

Histology FSH

0.7-11 11.0-20.0 20.0-30.0 ≥30.0

Normal spermatogenesis 2 . . .

Hypospermatogenesis 5 3 1

Maturation arrest 10 7 7 .

Sertoli cell only 5 5 5 3

Fibrosis 1 1 . .

Inadequate

1

Total 23 17 13 3

The FSH levels were noted to be higher in patients with the Sertoli cell only pattern,

with a mean of 21.3mIU /ml as compared to those with maturation arrest (mean of

13.2mIU/ml). However, this difference was not statistically significant, (p=0.7).The

mean FSH among those with normal spermatogenesis was 6.5mIU/ml.

10. Tubule diameter and histology:-The Kruskal-Wallis test was used to determine

whether there was a correlation between tubule diameter and the various histological

patterns. The number of biopsies with fibrosis was only two, therefore this category

could not be included in thisanalysis. Hence, this test was done using the results of the

remaining 67 subjects.According to theKruskal-Wallis test , there was a significant

correlation between tubule diameter and histology (p= 0.008)

73

To further substantiate, a Post Hoc test was done. In this test, the average tubule

diametersin each histological pattern was compared with the others, as shown in the

figure below. The post Hoc test showed significant differences in tubule diameter in

only two of the five histological patterns, namely: normal spermatogenesis and Sertoli

cell only pattern (p=0.017). There was no significant correlation between the tubule

diameters of the other histological subtypes.

Fig.28. Statistical correlation between tubule diameter and histological subtypes

Normal spermatogenesis p = 0.017

Sertoli cell only Maturation arrest p = 0.3

Hypospermatogenesis p = 0.7

Sertoli cell only p= 0.017

Normal spermatogenesis Maturation arrest p = 0.5

Hypospermatogenesis p = 0.8

Maturation arrest and Hypospermatogenesis p= 1

6.Cytogenetic analysis:(Table 17)

Cytogenetic analysis of peripheral blood was done in 24patients and showed a

normal karyotype (46,XY) in all but one patient, including the one whose testicular

biopsy was inadequate for histological evaluation.

74

Only one patient, a 23-year-old male had an abnormal karyotype. This male patient

who had a normal phenotype had 45 chromosomes with loss of the Y chromosomeand

only one X chromosome – a 45,X male. This patient had a critically low testicular

volume of 8 ml; his serum FSH showed a normal value of 8.79mIU/ml.

The single patient who had an abnormal karyotype was a 23-year-old male. This

patient appeared phenotypically normal except for a (critically) low testicular volume

of 8 ml. Cytogenetic analysis showed 45 chromosomes only one sex chromosome, the

X chromosome. The Y chromosome was not present. This patient was therefore a

45,X male. One homologue of chromosome 1 showed an additional band at the

terminal end of its long (q) arm. In view of the presence of the testis, a detailed

cytogenetic analysis was done using FISH probes for the SRY gene on the short (p)

arm of chromosome Y , the centromeres of the X and Y chromosomes and the

heterochromatin region of the Y chromosome. Metaphase FISH analysis confirmed

that the SRY gene was added to the terminal end of chromosome 1q (qter). No other

Y chromosome material could be seen. The karyotype of this patient was therefore re-

written as 45,X, add (1)(q44)[25].ish add (1)(1pter1qter ::Yp11.2 Ypter) (1p36+,

1q44+, DXZ1+, SRY+, DYZ3-, DYZ1-).nucish SRY×1, DYZ3×0)[100],(DXZ1×1,

DYZ1×0)[100],(1p36, 1q44)×2[100]). The presence of the SRY gene conferred the

male phenotype even though his karyotype showed only one sex chromosome, the X

chromosome.

75

Table 17. Classification based on karyotype done on 24 patients.

Karyotype Number (%)

Normal - 46,XY 23(96)

Abnormal - 45,X male 1(4)

7. Y chromosome microdeletion study(Tables 18)

This was done in 24 patients , 21 of whom did not show deletions of the AZF a,b or c

loci. Three patients showed deletions. Two patients had deletions of the AZFb region

only. The third was the 45,X male who showed absence of all three AZF loci, as

expected, because these loci are present on the long arm of the Y chromosome,which

was absent in him.

Table 18. Classification based on Y chromosome microdeletion status

Histomorphological and cytogenetic correlation:- Testicular biopsy in both patients

with the AZFb microdeletion showed maturation arrest, one at the level of primary

spermatocytes and the other at the level of spermatogonia. Testicular biopsy of the

45,X male who had microdeletions involving AZF a, b and c loci, showed Sertoli

cell only morphology. Table 12. Comparison of histology,Ychromosome

microdeletion status and karyotype

Y chromosome microdeletion status Number (%)

Negative 21(88)

Positive

AZF b deletion 2(8)

AZF a,b,c deletion 1(4)

76

Table 19: Relation of Histology to karyotype and Y chromosome mcrodeletion in the

abnormal cases

Histology Y chromosome

microdeletion

Karyotype

Primary spermatocyte AZFb 46,XY

Spematogonia AZFb 46,XY

Sertoli cell only AZFa,b,c 45,X, der(1)t(Y;1)(p11.2;q44)

77

DISCUSSION:

Infertility is an important reason for an otherwise healthy couple to seek medical help,

as the desire to have a child of their own is always strong. Nearly 15-20% of Indian

married couples are evaluated for infertility. Causes for female infertility are often the

main focus particularly in our society. The male causes are often neglected or ignored

due to the social stigma associated with it. However, statistics proves that in nearly

30% of the cases, the cause of infertility rests with the male partner an in 10% both the

male and female have abnormalities.(127)

Male infertility could be due to obstructive and non obstructive causes. Obstruction

during the transit of the viable sperms from the seminiferous tubules, to reach the

ejaculate, causes azoospermia. This implies that normal sperms are present in the

testicular tissue.

The obstructive causes of azoospermia signify that there is no fault with process or

apparatus of spermatogenesis. However during the transit of the viable sperms from

the seminiferous tubules, there is a focus of blockage, preventing them from becoming

a part of the ejaculate. This implies that there is viable sperm in the testicular tissue to

work with, and all that is then required is their medical extraction. This holds promise

for the couple seeking help.

Among the non- obstructive causes, the problem could be varied. The range of causes

can be from anti-sperm antibodies to abnormalities with the intrinsic infrastructure or

the mechanism of spermatogenesis. The latter can due to the various histological

78

pictures described earlier in which the cellularity of not only the seminiferous tubules

but also of the supporting system can affect spermatogenesis.

We have described the histological features of the testis in males with azoospermia or

oligospermia, as well as the relevant clinical and laboaratory features .

It was noted that the men who underwent evaluation for infertility ranged from 23-47

years of age,with the maximum number being 31 to 35 years. The fact that older men

were being evaluated for infertility suggests that age is not as limiting a factor in

males as it is in females. Other reports have also noted that this appears to the most

common age group being investigated for infertility.(127)

Testicular volume may be investigated clinically, with a Prader orchidometer or

ultrasonographically. The orchidometer is a string of numbered medical

beadscommonly called the ‗Endocrine rosary‘. The beads are compared to the testis

and the number closest to it is read as the volume. Prepubertal sizes are 1–3 ml,

pubertal sizes are considered 4 ml and up and adult sizes are 12–25 ml. The estimation

of testicular volume by this method is considered effective, as it is nearly equal to the

result obtained ultrasonographically.(128)

As per literature, the testicular volume increases from puberty upto 30 years of age,

after which there is no relationship between age and testicular volume. (129)Our study

did not reveal any significant correlation between testicular volume and age. The

mean testicular volume among our patients who were less than 30 years of age was

12.5ml and mean testicular volume among our patients above 30 years of age, was the

same, being 12ml. Most of our patients in both groups had subnormal values of less

79

than 20ml, with the majority having critically low testicular volume(less than12ml).

As described earlier, a critically low testicular volume is an indication for testicular

biopsy, in azoospermia. With larger volumes(normal or subnormal), a less invasive

procedure such as needle aspiration, rather than microTESE or testicular biopsy, is the

preferred method to obtain sperms when there is obstructive azoospermia.

FSH levels were done in all 56 patients evaluated in our centre. but the results were

not available for those evaluated elsewhere. The mean FSH was high (15.5mIU/ml).

FSH was found to be elevated regardless of whether testicular volumes were 20 ml or

above, 12-15ml, or less than 12 ml. There was no statistically significant correlation

between FSH and testicular volumes (p=0.34). Further analysis was done to assess the

histology in those with normal and high FSH. It was found that the predominant

pattern seen in both groups was maturation arrest, which was the most common

histological finding overall. However, it was noted that in the group with high FSH

there were almost equal numbers of biopsies with Sertoli cells only (13) and

maturation arrest (14). Morever, in the three patients with very high levels of

FSH(>30mIU/ml), Sertoli cells only was their histological pattern.

The majority of our patients (88%) had abnormal testicular morphology. All the

histological patterns associated with infertility were seen in our series, namely

Sertoli cell only, maturation arrest, hypospermatogenesis,seminiferous tubule

hyalinization and normal spermatogenesis. Normal spermatogenesis was seen in 12%

of our patients which included three of the four with oligospermia. Among those with

abnormal morphology, the majority (40 %) fell under the category of maturation

80

arrest. This finding was similar to the report of a meta-analysis performed by

McLachlan et al of Austalia in 2012. The cohort of patients included those from

various centres across Australia and one centre in Denmark.(58).

Maturation arrest was further subclassified based on the level at which the arrest

occurred. Primary spermatocytes were the most common finding in most of our

biopsies in 23% among the total number of cases. On comparison of this parameter

with the study McLachlan et al, the percentage of cases with primary spermatocytes

was only 10.1%. This maybe attributed to the fact that his series had a significant

number of biopsies with mixed pattern, in which also the cases with primary

spermatocytes was 10%.

The next most prominent histological pattern was Sertoli cell only. This pattern was

accompained by Leydig cell hyperplasia. This group of patients had elevated levels of

FSH some of which were markedly high. This could explain the rise of testosterone in

these patients as well. There was observed a rise in testosterone in cases with high

FSH levels (130)

Sertoli cell only is a pattern that is usually associated with Klinefelter syndrome. (1)

However, none of our patients were phenotypically of this type.

It was interesting to note that three of our patients were noted to have granulomatous

inflammation on histological evaluation. This was the reason for the azoospermia.

However, it was also noted that in all three patients, normal spermatogenesis was also

found in the same biopsy. Therefore, the azoospermia was both treatable and

reversible. It was suggested that the aetiology may be tuberculosis, as this is the most

81

common cause for granulomatous inflammation in India. Serological tests were

negative for syphilis.

There was noted to be a significant correlation between histology and tubule

diameter(p=0.008) and there was a statistically significant significant difference in

the tubule diameters between th histological subtypes of normal spermatogenesis and

Sertoli cells only(p=0.017)

When we compared our findings to other studies from India, and other countries, such

as Egypt, Saudi Arabia and England, we noted that there was no distinct pattern that

could be delineated. This suggests that a variety of histological patterns may be

associated with male infertility.The most common histopathological abnormality

among the evaluated Egyptian patients was Sertoli cell only,(2) which was also the

case in the study conducted in Saudi Arabia.(131) On the other hand, the highest

percentage of patients, in the English cohort had a diagnosis of hypospermatogenesis.

(132)(Table 20)

Table 20: Testicular biopsies in infertile males: Comparison of the histological

patterns in our patients and those from other countries

Histology Current

N=69 Egypt N=50 Saudi N=230 Australia N=352

Normal

spermatogenesis 12 24 31 3.2

Hypospermatogenesis 16 8 13 32

Maturation arrest 40 28 11 42.6

Sertoli cell only 29 34 39.5 15.7

Fibrosis 3 6 5 0.7

82

Fig.34: Bar diagram showing the most prominent histological category in each study

as compared to the current study.

When our study was compared to other studies from across India, the findings

were again variable, with no distinct patterns being identified. In a study by Parikh et

al(2012)(127) the most prominent catergory was normal spermatogenesis, similar to

that of an earlier study from our institution. In the study by Purohit et al(2004),(133)

two categories were equally significant, namely Sertoli cells only and

hypospermaogenesis.

05

101520253035404550

current (N=69)

Egypt N=50

saudi N=230

Australia N=352

England N=100

83

Table 21: Testicular biopsies in infertile males : Comparison of thehistological

patterns in our patients and those from India

Fig. 35: Bar diagram showing most prominent histologic category in each Indian

study.

05

1015202530354045

current (N=69)

Parekh et al N=80

Purohit et al N=58

Manoj et el N=95

Histology Current

N=69

Parikh et al

N=80

Purohit et al

N=58

Our

institution n

1991

(unpublished)

Normal

spermatogenesis 12 36 16 38

Hypospermatogenesis 16 7.5 26 20

Maturation arrest 40 11.25 8 22

Sertoli cell only 29 18.75 26 12

Fibrosis 3 11.25 18 6.3

84

It was also observed, when comparison was made between the current study and

the previous study in our institution, that despite the similar set of patients being

evaluated in the same institution, the pattern of pathologic diagnosis was different at

different times.

It was speculated whether such a change in the spectrum of diagnoses in the country

would have any backing from literature. Hence we attempted to study the different

studies done by Saudi Arabia over different time periods within the same region of the

country, thus focussing on the same population. Three studies were undertaken in

2000, 2011 and 2012 by Al Raees et al,(131) Abdulla et al (134)and Madbouly et al

(135)respectively. The following were the comparative results.

Table 22: Histological profile- change in pattern over the years in Saudi Arabia.

Histology Al Raees et al

2000, n=230

Abdulla et al 2011

n=100

Madbouly et al

2012 , n=447

Normal

spermatogenesis 31 13 5.8

Hypospermatogenesis 13 29 36.5

Maturation arrest 11 12 13.7

Sertoli cell only 39.5 16 31.5

Fibrosis 5 16 10.5

85

Fig.36: Bar diagram showing the shift in histology over the years in Saudi Arabia

However, there are discrepancies in this comparison. The histopathological

classification was not strictly uniform among these three studies. In the study by

Abdullah et al(2011) there are two categories named mixed and discordant patterns

which are not present in the other two studies. Despite this, there is a similarity

between the studies of 2011 and 2012, the most common diagnosis in these being

hypospermatogenesis. The study done in 2000 is distinct with the most cases being

diagnosed as Sertoli cells only.

All these studies emphasise that infertility may be associated with a variety of

histological patterns. This implied that there was a change in the trend of histological

patterns in patients being evaluated for infertility.

There was noted to be an increase in FSH values in the category with the histological

diagnosis of Sertoli cell only. This finding has been supported by literature, implying

05

1015202530354045

2000 N=230

2011 N=100

2012 N=447

86

that an early biochemical investigation might be able to give a clue to the

histopathological diagnosis, although, this is not always the case.

Cytogenteic analysis was normal in 23 patient s. on patient was a male with 45

chromosome X with no visible y chromosome, Klinefelter Syndrome which is the

most common genetic abnormality of sexual development usually seen, but was not

found in our series of karyotypes.

The 45, X male: This is a very rare condition with fewer than 50 patients described till

date. The phenotype is male because of the presence of the SRY gene, the testis

determining factor. However, the rest of the Y chromosomes including its long arm,

which carries the AZF gene is absent, as a result of which there is no there is

azoospermia and infertility. This condition results due to an error in meiotic

recombination.

Y chromosome microdeletion studies are now an essential part of the work up of

infertlity as they help to determine which patients benefit from ART as described

earlier. We had two patients with AZFb , with normal karyotypes whose histology

showed maturation arrest . AZFb deletions have poor prognosis as sperms cannot be

retrieved from such patients, The patient with 45 X, as expected, had AZF a,b&c

deletions due to the absence of the Y chromosome, on which lies the AZF genes. This

patient showed only Sertloi cells on histology.. Some patients with these deletions

may benefit from assisted reproductive techniques. This test helps to identify these

patients.

87

CONCLUSIONS:

Testicular biopsies were adequate in 69/70 patients evaluated for azoospsermia/

oligospermia.

The majority of patients (44%) belonged to the 31-35 years age group.

Testicular volumes were critically low volumes (56%)(less than 12 ml) .

FSH level ranged from low normal to high (2.42-56.4mIu/ml) ,with 59%

having elevated FSH(>11mIU/ml), with an overall mean value of 15.5mIU/ml.

The highest levels were seen in those with Sertoli cell only pattern.

The most common histologic subtype was maturation arrest(40%), followed by

Sertoli cell only pattern(29%). Normal spermatogenesis was seen in 12%.

There was a statistically significant correlation between tubule diameter and

histology, only in two subgroups namely the Sertoli cell only and normal

spermatogenesis. The other histological subtypes did not show any correlation.

Three patients who were suspected to have tuberculosis, had granulomatous

orchitis, with no serological evidence of syphilis.

Karyotypes were normal in 23/24 patients analysed. The patient with the

abnormal karyotype was a 45,X male with the SRY gene present on

chromosome 1.

Y chromosome microdeletion studies were normal in 21/24 patients studied.

Two of the remaining three, showed AZF b deletion, the biopsies of both

showed maturation arrest. The third patient who was a 45, X male, had

deletions of AZF a, b and c, reflecting the absence of the long arm of the Y

chromosome; his testicular biopsy showed Sertoli cells only.

Both the patients with AZFb deletion had maturation arrest

88

LIMITATIONS:

All patients evaluated for infertility did not have all the serum hormone levels

assessed, namely, FSH, LH, testosterone and prolactin.

Genetic analysis had not been done in all patients who had biopsies.

89

BIBLIOGRAPHY:

1. Cerilli LA, Kuang W, Rogers D. A practical approach to testicular biopsy interpretation for male infertility.

Arch Pathol Lab Med. 2010;134(8):1197–1204.

2. Rashed MM, Ragab NM, Shalaby AR, Ragab WK. Patterns Of Testicular Histopathology In Men With

Primary Infertility. Internet J Urol [Internet]. 2007 Dec 31 [cited 2016 Jul 23];5(2). Available from:

http://ispub.com/IJU/5/2/8836

3. Pryor JL, Kent-First M, Muallem A, Van Bergen AH, Nolten WE, Meisner L, et al. Microdeletions in the Y

chromosome of infertile men. N Engl J Med. 1997;336(8):534–540.

4. de Kretser DM, Loveland KL, Meinhardt A, Simorangkir D, Wreford N. Spermatogenesis. Hum Reprod

Oxf Engl. 1998 Apr;13 Suppl 1:1–8.

5. PubMed entry [Internet]. [cited 2016 Jul 23]. Available from:

http://www.ncbi.nlm.nih.gov/pubmed/9914171

6. Cheng CY, Mruk DD. The biology of spermatogenesis: the past, present and future. Philos Trans R Soc

Lond B Biol Sci. 2010 May 27;365(1546):1459–63.

7. Plant TM, Ramaswamy S, Simorangkir D, Marshall GR. Postnatal and pubertal development of the rhesus

monkey (Macaca mulatta) testis. Ann N Y Acad Sci. 2005 Dec;1061:149–62.

8. Endocrine control of spermatogenesis Role of FSH and LH.pdf.

9. Parvinen M. Regulation of the seminiferous epithelium. Endocr Rev. 1982;3(4):404–17.

10. Bartlett JM, Kerr JB, Sharpe RM. The effect of selective destruction and regeneration of rat Leydig cells on

the intratesticular distribution of testosterone and morphology of the seminiferous epithelium. J Androl.

1986 Aug;7(4):240–53.

11. DEFINITION OF THE STAGES OF THE CYCLE OF THE SEMINIFEROUS EPITHELIUM IN THE

RAT - Leblond - 2006 - Annals of the New York Academy of Sciences - Wiley Online Library [Internet].

[cited 2016 Jul 23]. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1749-

6632.1952.tb26576.x/abstract

12. Mali P, Virtanen I, Parvinen M. Vimentin Expression in Spermatogenic and Sertoli Cells is Stage-related in

Rat Seminiferous Epithelium. Andrologia. 1987 Nov 12;19(6):644–53.

13. Hormonal control of mammalian spermatogenesis. | POPLINE.org [Internet]. [cited 2016 Jul 23]. Available

from: http://www.popline.org/node/485030

14. Tahka KM. Local control mechanisms in the testis. Int J Dev Biol. 2003 Feb 1;33(1):141–8.

15. Isomaa V, Parvinen M, Jänne OA, Bardin CW. Nuclear Androgen Receptors in Different Stages of the

Seminiferous Epithelial Cycle and the Interstitial Tissue of Rat Testis. Endocrinology. 1985 Jan

1;116(1):132–7.

16. Galdieri M, Zani B. Hormonal induced changes in sertoli cell glycoproteins. Cell Biol Int Rep. 1981

Feb;5(2):111.

17. Jegou B, Peake RA, Irby DC, de Kretser DM. Effects of the induction of experimental cryptorchidism and

subsequent orchidopexy on testicular function in immature rats. Biol Reprod. 1984 Feb;30(1):179–87.

18. Vihko KK, Suominen JJ, Parvinen M. Cellular regulation of plasminogen activator secretion during

spermatogenesis. Biol Reprod. 1984 Sep;31(2):383–9.

90

19. Le Magueresse B, Le Gac F, Loir M, Jégou B. Stimulation of rat Sertoli cell secretory activity in vitro by

germ cells and residual bodies. J Reprod Fertil. 1986 Jul;77(2):489–98.

20. Rivarola MA, Sanchez P, Saez JM. Stimulation of ribonucleic acid and deoxyribonucleic acid synthesis in

spermatogenic cells by their coculture with Sertoli cells. Endocrinology. 1985 Nov;117(5):1796–802.

21. Steinberger E. Hormonal control of mammalian spermatogenesis. Physiol Rev. 1971 Jan;51(1):1–22.

22. Sharpe RM. Paracrine control of the testis. Clin Endocrinol Metab. 1986 Feb;15(1):185–207.

23. Aoki A, Fawcett DW. Is there a local feedback from the seminiferous tubules affecting activity of the

Leydig cells? Biol Reprod. 1978 Aug;19(1):144–58.

24. Kerr JB, Donachie K. Regeneration of Leydig cells in unilaterally cryptorchid rats: evidence for stimulation

by local testicular factors. Cell Tissue Res. 1986;245(3):649–55.

25. Tähkä KM, Rajaniemi H. Photoperiodic modulation of testicular LH receptors in the bank vole

(Clethrionomys glareolus). J Reprod Fertil. 1985 Nov;75(2):513–9.

26. Huhtaniemi I, Bergh A, Nikula H, Damber JE. Differences in the regulation of steroidogenesis and tropic

hormone receptors between the scrotal and abdominal testes of unilaterally cryptorchid adult rats.

Endocrinology. 1984 Aug;115(2):550–5.

27. Bergh A. Paracrine regulation of Leydig cells by the seminiferous tubules. Int J Androl. 1983 Feb;6(1):57–

65.

28. Bergh A, Damber JE. Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term

cryptorchidism. Int J Androl. 1984 Oct;7(5):409–18.

29. Parvinen M, Nikula H, Huhtaniemi I. Influence of rat seminiferous tubules on Leydig cell testosterone

production in vitro. Mol Cell Endocrinol. 1984 Oct;37(3):331–6.

30. Syed V, Khan SA, Ritzen EM. Stage-specific inhibition of interstitial cell testosterone secretion by rat

seminiferous tubules in vitro. Mol Cell Endocrinol. 1985 May;40(2–3):257–64.

31. Tabone E, Benahmed M, Reventos J, Saez JM. Interactions between immature porcine Leydig and Sertoli

cells in vitro. An ultrastructural and biochemical study. Cell Tissue Res. 1984;237(2):357–62.

32. Verhoeven G, Cailleau J. A factor in spent media from Sertoli-cell-enriched cultures that stimulates

steroidogenesis in Leydig cells. Mol Cell Endocrinol. 1985 Apr;40(1):57–68.

33. Carreau S, Papadopoulos V, Drosdowsky MA. Stimulation of adult rat Leydig cell aromatase activity by a

Sertoli cell factor. Endocrinology. 1988 Mar;122(3):1103–9.

34. Hsueh AJ. Direct effects of gonadotropin releasing hormone on testicular Leydig cell functions. Ann N Y

Acad Sci. 1982;383:249–71.

35. Sharpe RM, Doogan DG, Cooper I. Intratesticular factors and testosterone secretion: the role of luteinizing

hormone in relation to changes during puberty and experimental cryptorchidism. Endocrinology. 1986

Nov;119(5):2089–96.

36. Hedger MP, Robertson DM, Browne CA, de Kretser DM. The isolation and measurement of luteinizing

hormone-releasing hormone (LHRH) from the rat testis. Mol Cell Endocrinol. 1985 Sep;42(2):163–74.

37. Nikula H, Huhtaniemi I. Gonadotropin-releasing hormone agonist activates protein kinase C in rat Leydig

cells. Mol Cell Endocrinol. 1988 Jan;55(1):53–9.

91

38. Sharpe RM, Doogan DG, Cooper I. Direct effects of a luteinizing hormone-releasing hormone agonist on

intratesticular levels of testosterone and interstitial fluid formation in intact male rats. Endocrinology. 1983

Oct;113(4):1306–13.

39. Sharpe RM, Doogan DG, Cooper I. Factors determining whether the direct effects of an LHRH agonist on

Leydig cell function in vivo are stimulatory or inhibitory. Mol Cell Endocrinol. 1983 Sep;32(1):57–71.

40. Damber JE, Bergh A, Daehlin L. Testicular blood flow, vascular permeability, and testosterone production

after stimulation of unilaterally cryptorchid adult rats with human chorionic gonadotropin. Endocrinology.

1985 Nov;117(5):1906–13.

41. Hedger MP, Robertson DM, Tepe SJ, Browne CA, de Kretser DM. Degradation of luteinizing hormone-

releasing hormone (LHRH) and an LHRH agonist by the rat testis. Mol Cell Endocrinol. 1986

Jun;46(1):59–70.

42. Clayton RN, Detta A, Naik SI, Young LS, Charlton HM. Gonadotrophin releasing hormone receptor

regulation in relationship to gonadotrophin secretion. J Steroid Biochem. 1985 Nov;23(5B):691–702.

43. Huhtaniemi IT, Yamamoto M, Ranta T, Jalkanen J, Jaffe RB. Follicle-stimulating hormone receptors

appear earlier in the primate fetal testis than in the ovary. J Clin Endocrinol Metab. 1987 Dec;65(6):1210–

4.

44. Rommerts FF, Themmen AP. LHRH, the role of LHRH (agonists) in the regulation of gonadal function.

Acta Endocrinol Suppl (Copenh). 1986;276:76–84.

45. Parvinen M, Nikula H, Huhtaniemi I. Influence of rat seminiferous tubules on Leydig cell testosterone

production in vitro. Mol Cell Endocrinol. 1984 Oct;37(3):331–6.

46. Sharpe RM, Bartlett JM. Intratesticular distribution of testosterone in rats and the relationship to the

concentrations of a peptide that stimulates testosterone secretion. J Reprod Fertil. 1985 May;74(1):223–36.

47. Boitani C, Ritzén EM, Parvinen M. Inhibition of rat Sertoli cell aromatase by factor(s) secreted specifically

at spermatogenic stages VII and VIII. Mol Cell Endocrinol. 1981 Jul;23(1):11–22.

48. Papadopoulos V, Kamtchouing P, Drosdowsky MA, Hochereau de Reviers MT, Carreau S. Adult rat

Sertoli cells secrete a factor or factors which modulate Leydig cell function. J Endocrinol. 1987

Sep;114(3):459–67.

49. Kalla NR, Zarabi S. Presence of luteinizing hormone receptor binding inhibitor (LH-RBI) in ovine testis.

Andrologia. 1982 Jun;14(3):265–9.

50. Pandey KN, Maki M, Inagami T. Detection of renin mRNA in mouse testis by hybridization with renin

cDNA probe. Biochem Biophys Res Commun. 1984 Dec 14;125(2):662–7.

51. Bardin CW, Morris PL, Chen CL, Boitani C, Krieger DT. The action of pro-opiomelanocortin-derived

peptides on testicular cells. Ann N Y Acad Sci. 1987;512:308–17.

52. Douglass J, Cox B, Quinn B, Civelli O, Herbert E. Expression of the prodynorphin gene in male and female

mammalian reproductive tissues. Endocrinology. 1987 Feb;120(2):707–13.

53. Yoon DJ, Sklar C, David R. Presence of immunoreactive corticotropin-releasing factor in rat testis.

Endocrinology. 1988 Feb;122(2):759–61.

54. Tähkä KM. Current aspects of Leydig cell function and its regulation. J Reprod Fertil. 1986

Nov;78(2):367–80.

55. Yaman O, Ozdiler E, Seçkiner I, Göğüş O. Significance of serum FSH levels and testicular morphology in

infertile males. Int Urol Nephrol. 1999;31(4):519–23.

92

56. Johnsen SG. Testicular biopsy score count--a method for registration of spermatogenesis in human testes:

normal values and results in 335 hypogonadal males. Hormones. 1970;1(1):2–25.

57. testicular hp 2.pdf.

58. McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM, Skakkebaek NE. Histological

evaluation of the human testis--approaches to optimizing the clinical value of the assessment: Mini Review.

Hum Reprod. 2006 Oct 10;22(1):2–16.

59. testicular hp 1.pdf.

60. De Braekeleer M, Dao TN. Cytogenetic studies in male infertility: a review. Hum Reprod Oxf Engl. 1991

Feb;6(2):245–50.

61. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, et al. Diverse spermatogenic defects in

humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet.

1995 Aug;10(4):383–93.

62. Reijo R, Alagappan RK, Patrizio P, Page DC. Severe oligozoospermia resulting from deletions of

azoospermia factor gene on Y chromosome. Lancet Lond Engl. 1996 May 11;347(9011):1290–3.

63. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome

azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet. 1996 Jul;5(7):933–

43.

64. Brandell RA, Mielnik A, Liotta D, Ye Z, Veeck LL, Palermo GD, et al. AZFb deletions predict the absence

of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum

Reprod Oxf Engl. 1998 Oct;13(1O):2812–5.

65. Martínez MC, Bernabé MJ, Gómez E, Ballesteros A, Landeras J, Glover G, et al. Screening for AZF

deletion in a large series of severely impaired spermatogenesis patients. J Androl. 2000 Oct;21(5):651–5.

66. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of

the human Y chromosome long arm. Hum Genet. 1976 Oct 28;34(2):119–24.

67. Hartung M, Devictor M, Codaccioni JL, Stahl A. Yq deletion and failure of spermatogenesis. Ann

Génétique. 1988;31(1):21–6.

68. Chandley AC, Gosden JR, Hargreave TB, Spowart G, Speed RM, McBeath S. Deleted Yq in the sterile son

of a man with a satellited Y chromosome (Yqs). J Med Genet. 1989 Mar;26(3):145–53.

69. Andersson M, Page DC, Pettay D, Subrt I, Turleau C, de Grouchy J, et al. Y;autosome translocations and

mosaicism in the aetiology of 45,X maleness: assignment of fertility factor to distal Yq11. Hum Genet.

1988 May;79(1):2–7.

70. Bardoni B, Zuffardi O, Guioli S, Ballabio A, Simi P, Cavalli P, et al. A deletion map of the human Yq11

region: implications for the evolution of the Y chromosome and tentative mapping of a locus involved in

spermatogenesis. Genomics. 1991 Oct;11(2):443–51.

71. Ma K, Sharkey A, Kirsch S, Vogt P, Keil R, Hargreave TB, et al. Towards the molecular localisation of the

AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the

human Y chromosome. Hum Mol Genet. 1992 Apr;1(1):29–33.

72. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, et al. The male-specific

region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003 Jun

19;423(6942):825–37.

93

73. Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, et al. The AZFc

region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

Nat Genet. 2001 Nov;29(3):279–86.

74. Yen P. The fragility of fertility. Nat Genet. 2001 Nov;29(3):243–4.

75. Repping S, Skaletsky H, Brown L, van Daalen SKM, Korver CM, Pyntikova T, et al. Polymorphism for a

1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and

haploid selection. Nat Genet. 2003 Nov;35(3):247–51.

76. Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, et al. Recombination between

palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure.

Am J Hum Genet. 2002 Oct;71(4):906–22.

77. Blanco P, Shlumukova M, Sargent CA, Jobling MA, Affara N, Hurles ME. Divergent outcomes of

intrachromosomal recombination on the human Y chromosome: male infertility and recurrent

polymorphism. J Med Genet. 2000 Oct;37(10):752–8.

78. Kamp C, Hirschmann P, Voss H, Huellen K, Vogt PH. Two long homologous retroviral sequence blocks in

proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination events. Hum

Mol Genet. 2000 Oct 12;9(17):2563–72.

79. Sun C, Skaletsky H, Rozen S, Gromoll J, Nieschlag E, Oates R, et al. Deletion of azoospermia factor a

(AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses. Hum Mol

Genet. 2000 Sep 22;9(15):2291–6.

80. Kamp C, Huellen K, Fernandes S, Sousa M, Schlegel PN, Mielnik A, et al. High deletion frequency of the

complete AZFa sequence in men with Sertoli-cell-only syndrome. Mol Hum Reprod. 2001 Oct;7(10):987–

94.

81. Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, et al. Recombination between

palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure.

Am J Hum Genet. 2002 Oct;71(4):906–22.

82. Lange J, Skaletsky H, van Daalen SKM, Embry SL, Korver CM, Brown LG, et al. Isodicentric Y

chromosomes and sex disorders as byproducts of homologous recombination that maintains palindromes.

Cell. 2009 Sep 4;138(5):855–69.

83. Krausz C, Chianese C, Giachini C, Guarducci E, Laface I, Forti G. The Y chromosome-linked copy number

variations and male fertility. J Endocrinol Invest. 2011 May;34(5):376–82.

84. Sin H-S, Koh E, Shigehara K, Sugimoto K, Maeda Y, Yoshida A, et al. Features of constitutive gr/gr

deletion in a Japanese population. Hum Reprod Oxf Engl. 2010 Sep;25(9):2396–403.

85. Yang Y, Ma M, Li L, Su D, Chen P, Ma Y, et al. Differential effect of specific gr/gr deletion subtypes on

spermatogenesis in the Chinese Han population. Int J Androl. 2010 Oct 1;33(5):745–54.

86. Fernandes S, Huellen K, Goncalves J, Dukal H, Zeisler J, Rajpert De Meyts E, et al. High frequency of

DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia. Mol Hum Reprod. 2002

Mar;8(3):286–98.

87. Ferlin A, Tessari A, Ganz F, Marchina E, Barlati S, Garolla A, et al. Association of partial AZFc region

deletions with spermatogenic impairment and male infertility. J Med Genet. 2005 Mar;42(3):209–13.

88. Giachini C, Guarducci E, Longepied G, Degl‘Innocenti S, Becherini L, Forti G, et al. The gr/gr deletion(s):

a new genetic test in male infertility? J Med Genet. 2005 Jun;42(6):497–502.

94

89. Krausz C, Giachini C, Xue Y, O‘Bryan MK, Gromoll J, Rajpert-de Meyts E, et al. Phenotypic variation

within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal

background. J Med Genet. 2009 Jan;46(1):21–31.

90. Choi J, Song S-H, Bak CW, Sung SR, Yoon TK, Lee DR, et al. Impaired spermatogenesis and gr/gr

deletions related to Y chromosome haplogroups in Korean men. PloS One. 2012;7(8):e43550.

91. Visser L, Westerveld GH, Korver CM, van Daalen SKM, Hovingh SE, Rozen S, et al. Y chromosome gr/gr

deletions are a risk factor for low semen quality. Hum Reprod Oxf Engl. 2009 Oct;24(10):2667–73.

92. Yang Y, Xiao C-Y, A Z-C, Zhang S-Z, Li X, Zhang S-X. DAZ1/DAZ2 cluster deletion mediated by gr/gr

recombination per se may not be sufficient for spermatogenesis impairment: a study of Chinese

normozoospermic men. Asian J Androl. 2006 Mar;8(2):183–7.

93. Ferlin A, Moro E, Onisto M, Toscano E, Bettella A, Foresta C. Absence of testicular DAZ gene expression

in idiopathic severe testiculopathies. Hum Reprod Oxf Engl. 1999 Sep;14(9):2286–92.

94. Foresta C, Moro E, Rossi A, Rossato M, Garolla A, Ferlin A. Role of the AZFa candidate genes in male

infertility. J Endocrinol Invest. 2000 Nov;23(10):646–51.

95. Silber SJ, Alagappan R, Brown LG, Page DC. Y chromosome deletions in azoospermic and severely

oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction. Hum

Reprod Oxf Engl. 1998 Dec;13(12):3332–7.

96. Sun C, Skaletsky H, Birren B, Devon K, Tang Z, Silber S, et al. An azoospermic man with a de novo point

mutation in the Y-chromosomal gene USP9Y. Nat Genet. 1999 Dec;23(4):429–32.

97. Krausz C, Quintana-Murci L, Barbaux S, Siffroi J-P, Rouba H, Delafontaine D, et al. A High Frequency of

Y Chromosome Deletions in Males with Nonidiopathic Infertility 1. J Clin Endocrinol Metab.

1999;84(10):3606–3612.

98. Krausz C, Rajpert-De Meyts E, Frydelund-Larsen L, Quintana-Murci L, McElreavey K, Skakkebaek NE.

Double-blind Y chromosome microdeletion analysis in men with known sperm parameters and

reproductive hormone profiles: microdeletions are specific for spermatogenic failure. J Clin Endocrinol

Metab. 2001 Jun;86(6):2638–42.

99. Simoni M, Tüttelmann F, Gromoll J, Nieschlag E. Clinical consequences of microdeletions of the Y

chromosome: the extended Münster experience. Reprod Biomed Online. 2008 Feb;16(2):289–303.

100. Tyler-Smith C, Krausz C. The will-o‘-the-wisp of genetics--hunting for the azoospermia factor gene. N

Engl J Med. 2009 Feb 26;360(9):925–7.

101. Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility. Int J Androl. 2003

Apr;26(2):70–5.

102. Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis: what is the clinical

prognostic value of Y chromosome microdeletion analysis? Hum Reprod Oxf Engl. 2000 Jul;15(7):1431–4.

103. Hopps CV. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc

regions. Hum Reprod. 2003 Aug 1;18(8):1660–5.

104. Kleiman SE, Almog R, Yogev L, Hauser R, Lehavi O, Paz G, et al. Screening for partial AZFa

microdeletions in the Y chromosome of infertile men: is it of clinical relevance? Fertil Steril. 2012

Jul;98(1):43–7.

105. Kleiman SE, Yogev L, Lehavi O, Hauser R, Botchan A, Paz G, et al. The likelihood of finding mature

sperm cells in men with AZFb or AZFb-c deletions: six new cases and a review of the literature (1994-

2010). Fertil Steril. 2011 May;95(6):2005–12, 2012-4.

95

106. Longepied G, Saut N, Aknin-Seifer I, Levy R, Frances A-M, Metzler-Guillemain C, et al. Complete

deletion of the AZFb interval from the Y chromosome in an oligozoospermic man. Hum Reprod Oxf Engl.

2010 Oct;25(10):2655–63.

107. Soares AR, Costa P, Silva J, Sousa M, Barros A, Fernandes S. AZFb microdeletions and oligozoospermia--

which mechanisms? Fertil Steril. 2012 Apr;97(4):858–63.

108. Luetjens CM, Gromoll J, Engelhardt M, Von Eckardstein S, Bergmann M, Nieschlag E, et al.

Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and

immunohistochemical evaluation. Hum Reprod Oxf Engl. 2002 Sep;17(9):2258–66.

109. Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men

with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum

Reprod Oxf Engl. 2002 Nov;17(11):2813–24.

110. Kühnert B, Gromoll J, Kostova E, Tschanter P, Luetjens CM, Simoni M, et al. Case report: natural

transmission of an AZFc Y-chromosomal microdeletion from father to his sons. Hum Reprod Oxf Engl.

2004 Apr;19(4):886–8.

111. Kent-First MG, Kol S, Muallem A, Ofir R, Manor D, Blazer S, et al. The incidence and possible relevance

of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers.

Mol Hum Reprod. 1996 Dec;2(12):943–50.

112. Mulhall JP, Reijo R, Alagappan R, Brown L, Page D, Carson R, et al. Azoospermic men with deletion of

the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic

development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic

sperm injection. Hum Reprod Oxf Engl. 1997 Mar;12(3):503–8.

113. Kamischke A, Gromoll J, Simoni M, Behre HM, Nieschlag E. Transmission of a Y chromosomal deletion

involving the deleted in azoospermia (DAZ) and chromodomain (CDY1) genes from father to son through

intracytoplasmic sperm injection: case report. Hum Reprod Oxf Engl. 1999 Sep;14(9):2320–2.

114. Jiang MC, Lien YR, Chen SU, Ko TM, Ho HN, Yang YS. Transmission of de novo mutations of the

deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm

injection. Fertil Steril. 1999 Jun;71(6):1029–32.

115. Kleiman SE, Yogev L, Gamzu R, Hauser R, Botchan A, Paz G, et al. Three-generation evaluation of Y-

chromosome microdeletion. J Androl. 1999 Jun;20(3):394–8.

116. Page DC, Silber S, Brown LG. Men with infertility caused by AZFc deletion can produce sons by

intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum Reprod Oxf

Engl. 1999 Jul;14(7):1722–6.

117. Cram DS, Ma K, Bhasin S, Arias J, Pandjaitan M, Chu B, et al. Y chromosome analysis of infertile men

and their sons conceived through intracytoplasmic sperm injection: vertical transmission of deletions and

rarity of de novo deletions. Fertil Steril. 2000 Nov;74(5):909–15.

118. van Golde RJ, Wetzels AM, de Graaf R, Tuerlings JH, Braat DD, Kremer JA. Decreased fertilization rate

and embryo quality after ICSI in oligozoospermic men with microdeletions in the azoospermia factor c

region of the Y chromosome. Hum Reprod Oxf Engl. 2001 Feb;16(2):289–92.

119. Peterlin B, Kunej T, Sinkovec J, Gligorievska N, Zorn B. Screening for Y chromosome microdeletions in

226 Slovenian subfertile men. Hum Reprod Oxf Engl. 2002 Jan;17(1):17–24.

120. Lo Giacco D, Chianese C, Sánchez-Curbelo J, Bassas L, Ruiz P, Rajmil O, et al. Clinical relevance of Y-

linked CNV screening in male infertility: new insights based on the 8-year experience of a diagnostic

genetic laboratory. Eur J Hum Genet EJHG. 2014 Jun;22(6):754–61.

121. MoRITA Y. Histological Investigation of Testis in Infertile Man: Part I. Some Clinical Problems on

Testicular Biopsy. Nagoya J Med Sci. 1971;34(2):101–112.

96

122. Dym M. Basement Membrane Regulation of Sertoli Cells. Grantome [Internet]. [cited 2016 Sep 22];

Available from: http://grantome.com/grant/NIH/R01-HD016260-16

123. Krausz C, Hoefsloot L, Simoni M, Tüttelmann F. EAA/EMQN best practice guidelines for molecular

diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013. Andrology. 2014 Jan;2(1):5–19.

124. Korf BR. Overview of clinical cytogenetics. Curr Protoc Hum Genet Editor Board Jonathan Haines Al.

2001 Aug;Chapter 8:Unit 8.1.

125. Knoll JHM, Lichter P. In situ hybridization to metaphase chromosomes and interphase nuclei. Curr Protoc

Hum Genet Editor Board Jonathan Haines Al. 2005 May;Chapter 4:Unit 4.3.

126. 93643.pdf [Internet]. [cited 2016 Sep 18]. Available from: http://media.axon.es/pdf/93643.pdf

127. Testicular Biopsy in Male Infertility.pdf.

128. Sotos JF, Tokar NJ. Testicular volumes revisited: A proposal for a simple clinical method that can closely

match the volumes obtained by ultrasound and its clinical application. Int J Pediatr Endocrinol.

2012;2012(1):17.

129. Condorelli R, Calogero AE, La Vignera S. Relationship between Testicular Volume and Conventional or

Nonconventional Sperm Parameters. Int J Endocrinol. 2013 Sep 5;2013:e145792.

130. Shimon I, Lubina A, Gorfine M, Ilany J. Feedback Inhibition of Gonadotropins by Testosterone in Men

With Hypogonadotropic Hypogonadism: Comparison to the Intact Pituitary-Testicular Axis in Primary

Hypogonadism. J Androl. 2006 May 6;27(3):358–64.

131. Al-Rayess MM, Al-Rikabi AC. Morphologic patterns of male infertility in Saudi patients. A University

Hospital experience. Saudi Med J. 2000 Jul;21(7):625–8.

132. Meinhard E, McRae CU, Chisholm GD. Testicular Biopsy in Evaluation of Male Infertility. Br Med J.

1973 Sep 15;3(5880):577–81.

133. Purohit TM, Purohit MB, Dabhi BJ. Study of semen analysis and testicular biopsy in infertile male. Indian

J Pathol Microbiol. 2004 Oct;47(4):486–90.

134. Abdullah L, Bondagji N. Histopathological patterns of testicular biopsy in male infertility: A retrospective

study from a tertiary care center in the western part of Saudi Arabia. Urol Ann. 2011;3(1):19–23.

135. Madbouly K, Al-hooti Q, Albkri A, Ragheb S, Alghamdi K, Al-Jasser A. Clinical, endocrinological and

histopathological patterns of infertile Saudi men subjected to testicular biopsy: A retrospective study from a

single center. Urol Ann. 2012;4(3):166–71.

97

98

99

APPENDIX - 1

PROFILE OF PATIENTS ACCORDING TO HISTOLOGY:

UID age test

vol FSH karyotype Y chr

25 38 . . . .

40 31 . . . .

48 33 . . . .

52 30 . . . .

54 26 . 10.7 . .

56 33 . . . .

66 33 . 2.22 . .

70 35 . . . .

Profile of patients with normal spermatogenesis

UID Age Test

vol Fsh Karyotype Y chr

10 33 13.5 6.2 . .

12 29 . . . .

13 31 12 7.4 . .

15 43 17 22.8 . .

19 38 . . . .

22 31 15 6.83 . .

26 29 10 16.6 . .

32 36 20 6.46 . .

37 29 15 8.46 46, XY Negative

49 41 . 14.2 46, XY Negative

63 35 12 15.8 46, XY Negative

Profile of patients with hypospermatogenesis

100

ID Age Test

vol FSH Level of arrest karyotype

Y chr

deln

4 29 . . primary spermatocyte . .

5 29 20 28.6 primary spermatocyte . .

9 34 13 13.7 primary spermatocyte 46XY[40] negative


14 35 15 2.58 primary spermatocyte 46XY[20] AZFb


17 33 1.5 2.38 primary spermatocyte 46XY[20] negative

20 36 7.5 4.86 Spermatogonia 46XY[20] AZFb

21 28 . . Spermatogonia . .





35 37 10 14.7 secondary

spermatocyte 46XY[20] negative

36 39 . 3.02 primary spermatocyte . .


41 30 15 21 primary spermatocyte 46XY[25] negative

42 34 . 21.6 primary spermatocyte . .

46 36 10 25.1 spermatogonia . negative


50 36 10 11 spermatogonia . .

51 45 15 13.2 spermatogonia . .

57 33 10 16 primary spermatocyte . .

60 32 13 4.65 spermatogonia 46XY[20] negative

62 35 . . secondary

spermatocyte . .

64 26 . . primary spermatocyte . .

67 35 . 4.85 secondary

spermatocyte 46XY[20] negative

68 31 9 18.3 spermatogonia 46XY[20] negative

Profile of patients with maturation arrest

101

UID age

test


1 27 . . 1 .

2 29 20 13.5 46XY[20] negative

3 33 . 44.5 . .

6 32 12.5 14.5 46XY[20] negative

7 25 10 9.96 46XY[20] negative

8 41 10 10.8 . .

18 31 . 35 . .

23 35 12 5.13 . .

31 37 12 23.6 . .

34 32 15 18.6 46XY[20] negative

38 37 8 15.2 46XY[45] negative

43 33 10 8.97 . .

45 47 10 27.3 46XY[20] negative

53 31 . 26.6 46XY[20] negative

55 30 . . . .

58 31 . 26.6 46XY[20] negative

59 43 15 56.4 . .

61 23 8 8.79 45X[25] AZFabc

65 39 . 15.5 . .

69 31 10 26.6 46XY[20] negative

Profile of patients with Sertoli cell only

UID age

test


29 37 . 10.2 . .

44 29 9 15.3 . .

Profile of patients with peritubular fibrosis/ atrophic tubules.

102

APPENDIX - 2

PROFORMA

Name: Age:

Hospital no.:

Testicular volume:

Gonadal anomalies if any:

Semen analysis:

Hormonal status:-

FSH: LH:

Prolactin: Testosterone:

Histopathological diagnosis:

103

APPENDIX – 3

DATA SHEET

V2

un

iqu

e ID

ho

sp n

o.

bio

psy

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ete

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tub

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no

rmal

1.

neg

ativ

eM

atu

rati

on

arr

est

4

47

13

01

05

01

16

85

62

17

c4

69

/13

26

92

5.1

57

23

52

pri

mar

y sp

erm

ato

cyte

2n

orm

al1

..

Mat

ura

tio

n a

rres

t4

48

90

18

94

6o

uts

ide

13

45

/13

33

..

24

32

60

1n

orm

al s

per

mat

oge

nes

is2

no

rmal

1.

.n

orm

al s

per

mat

oge

nes

is3

49

13

00

70

04

66

14

91

97

f4

43

5/1

34

1.

14

.26

21

85

1o

ccas

ion

al s

per

mat

ozo

a1

no

rmal

14

6X

Y[2

0]

neg

ativ

eH

ypo

sp

erm

ato

gen

esis

5

50

13

03

08

02

69

40

22

86

f8

33

4/1

33

61

01

17

71

90

2sp

erm

ato

gon

ia2

no

rmal

1.

.M

atu

rati

on

arr

est

4

51

13

03

20

02

60

50

59

51

f9

78

7/1

34

51

51

3.2

22

52

50

2sp

erm

ato

gon

ia2

lym

ph

ocy

tes

1.

.M

atu

rati

on

arr

est

4

52

90

19

35

3o

uts

ide

12

92

2/1

33

0.

.9

02

75

1n

orm

al s

per

mat

oge

nes

is2

no

rmal

1.

.n

orm

al s

per

mat

oge

nes

is3

53

14

08

28

01

59

84

72

49

f3

25

57

/14

31

.2

6.6

86

17

92

nil

2n

orm

al1

46

XY[

20

]n

egat

ive

sc1

54

14

06

11

01

93

79

23

85

f2

17

66

/14

26

.1

0.7

64

39

71

no

rmal

sp

erm

ato

gen

esis

1ly

mp

ho

cyte

s an

d g

ran

ulo

mas1

..

no

rmal

sp

erm

ato

gen

esis

3

55

90

21

20

7o

uts

ide

13

80

8/1

43

0.

.1

21

18

01

nil

2n

orm

al1

..

sc1

56

90

28

97

ou

tsid

e6

94

9/1

43

3.

.6

42

02

1n

orm

al s

per

mat

oge

nes

is2

lym

ph

oh

isti

cyti

c ag

greg

ate

s1.

.n

orm

al s

per

mat

oge

nes

is3

57

14

05

23

02

77

84

56

42

f1

92

52

/14

33

10

16

12

61

90

2p

rim

ary

sper

mat

ocy

te2

no

rmal

1.

.M

atu

rati

on

arr

est

4

58

14

08

28

01

59

84

72

49

f3

25

57

/14

31

.2

6.6

10

82

10

2n

il2

no

rmal

14

6X

Y[2

0]

neg

ativ

esc

1

59

12

04

11

02

60

04

07

71

c1

19

43

/12

43

15

56

.41

81

54

2n

il2

no

rmal

1.

.sc

1

60

11

06

29

02

05

35

89

26

b2

02

71

/11

32

13

4.6

57

12

00

1sp

erm

ato

gon

ia2

no

rmal

14

6X

Y[2

0]

neg

ativ

eM

atu

rati

on

arr

est

4

61

14

07

08

03

28

89

09

60

f2

54

88

/14

23

88

.79

17

82

10

2n

il2

abse

nt

14

5X

[25

]A

ZFab

csc

1

62

90

22

25

3o

uts

ide

44

17

5/1

43

5.

.9

23

29

1se

con

dar

y sp

erm

ato

cyte

1n

om

al1

..

Mat

ura

tio

n a

rres

t4

63

14

07

15

03

30

82

56

92

f2

64

97

/14

35

12

15

.89

02

40

1sp

erm

atid

2Le

ydig

cel

l clu

ste

rs1

46

XY[

25

]n

egat

ive

Hyp

o s

per

mat

oge

nes

is5

64

90

19

60

1o

uts

ide

19

84

5/1

32

6.

.2

80

13

01

pri

mar

y sp

erm

ato

cyte

2Le

ydig

cel

l an

d in

flam

mat

ory

cel

ls1

..

Mat

ura

tio

n a

rres

t4

65

14

10

14

01

57

38

69

15

f3

91

06

/14

39

.1

5.5

32

15

02

nil

2n

orm

al1

..

sc1

66

12

05

07

03

90

94

15

24

d1

51

23

/12

33

.2

.22

50

02

60

1n

orm

al s

per

mat

oge

nes

is1

fore

ign

bo

dy

gian

t ce

ll re

acti

on

1.

.n

orm

al s

per

mat

oge

nes

is3

67

14

11

12

04

12

86

98

58

f4

32

43

/14

35

.4

.85

30

27

01

seco

nd

ary

sper

mat

ocy

te2

no

rmal

14

6X

Y[2

0]

neg

ativ

eM

atu

rati

on

arr

est

4

68

14

09

23

03

12

03

41

16

g3

61

65

/14

31

91

8.3

10

11

50

1sp

erm

ato

gon

ia2

no

rmal

14

6X

Y[2

0]

neg

ativ

eM

atu

rati

on

arr

est

4

69

14

05

16

03

50

84

72

49

f1

83

30

/14

31

10

26

.64

21

01

nil

1n

orm

al1

46

XY[

20

]n

egat

ive

sc1

70

90

22

34

8o

uts

ide

47

93

0/1

43

5.

.5

52

08

1n

orm

al s

per

mat

oge

nes

is1

no

rmal

1.

.n

orm

al s

per

mat

oge

nes

is3

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