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.
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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.
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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.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 Christian Medical College,
Vellore.
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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 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 Christian Medical College,
Vellore.
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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
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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
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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)
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http://clinicalgate.com/tag/harrisons-principles-of-internal-medicine-
Fig5. Paracrine control of spermatogenesis
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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
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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,
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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
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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.
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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
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(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)
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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
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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.
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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
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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
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Fig 8: Hypospermatoenesis http://www.archivesofpathology.org (1)
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.
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Fig. 9: Atrophy and peritubular fibrosis http://www.archivesofpathology.org (1)
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
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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
http://www.archivesofpathology.org (1)
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Fig. 11 ALGORITHM FOR EVALUATION OF TESTICULAR BIOPSY IN INFERTILITY
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Y CHROMOSOME MICRODELETION
Fig.12: Schematic representation of Y chromosome
Fig. 13: Representation of the cytological bands of the Y chromosome.
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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-
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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.
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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
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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).
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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
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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.
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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.
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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
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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)
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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.
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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.
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*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,
