By Bahram Robert Oliai, M.D.
By providing key information on the state of the testicular parenchyma and the potential reversibility of certain abnormalities, testicular biopsy can be of significant value in the clinical workup of infertile patients. In my previous position as a community hospital staff pathologist, on occasion I would be called upon to examine testicular biopsies, but their relative rarity and the paucity of clear guidelines for examination of these specimens tended to engender a fair amount of anxiety. This month I review this interesting and at times confusing topic, describing a methodical approach that provides useful practical information, in the hopes that my pathologist and urologist colleagues will find it helpful as they care for infertile patients. Detailed descriptions of pediatric testicular biopsy (for example in the workup of ambiguous genitalia) and semen analysis are beyond the scope of this review.
Normal histology of the testis
Before we embark on our journey to understand the pathology of the infertile testis, it is useful to review its normal microscopic anatomy. As we know, the normal adult testis is a paired organ, which lies in the scrotum suspended by the spermatic cord containing the vas deferens, blood vessels, and lymphatics that supply the organ. Generally each testis weighs between 15-19 g with the right being slightly heavier than the left. The testis is invested by three layers: The outer serosa or tunica vaginalis (covered by mesothelial cells), the tunica albuginea, and the inner tunica vasculosa. At the posterior aspect of the testis lies the “mediastinum testis” containing blood vessels, nerves, and a portion of a rete testis. The parenchyma is divided into lobules (approximately 250), each containing around 3-4 seminiferous tubules.
The testicular parenchyma is divided into interstitium and seminiferous tubules. The interstitium, accounting for 25 to 30% of the testicular mass, is composed of vessels, nerves, macrophages, mast cells, and the testosterone secreting Leydig cells.
Leydig cells are characterized by round vesicular nuclei containing 1-2 prominent nucleoli and abundant granular pink cytoplasm, sometimes containing pigment and so called Reinke crystalloids (FIG 2). The seminiferous tubules contain Sertoli cells and maturing germ cells and are bounded by a basement membrane. Sertoli cells usually comprise 10-15% of tubular cellular elements and lie at the tubular basement membrane. They embrace maturing germ cells with cytoplasmic extensions, supporting their development, like a caring mother looking after her young. Sertoli cells often have a somewhat triangular shape with basally oriented convoluted nuclei showing prominent nucleoli and frothy pink cytoplasm, sometimes containing lipid vacuoles, allowing for easy separation from the surrounding germ cells (FIG 3).
The rete testes, composed of complex tubules, receives the contents of the seminiferous tubules, emptying into the ductuli efferentes leading into the ciliated epididymis, and finally out into the vas deferens.
As a rule of thumb the size of an adequate testicular biopsy for infertility should be about the size of a grain of rice (3 mm) in any one dimension, and will contain 3-5 lobules, or 9-20 seminiferous tubules with intervening septa (which should not be over interpreted as fibrosis, FIG 1). One sample per testis is usually adequate, but it may be necessary to obtain bilateral specimens as the nature and/or stage of various lesions may differ from one testis to the other. This sample can be obtained from the portion opposite the rete testes via a 4-5 mm incision in the tunica albuginea. This incision will cause a small herniation of the parenchyma, allowing easy access to the testis.
Goals of biopsy
Ultimately the goal is to recognize which (if any) pattern of abnormality is present, quantify the various patterns present (much like we do when reporting a mixed germ cell tumor), and comment on any additional pathology present. The four main patterns of abnormality in the infertile testis include Sertoli only/pure germ cell aplasia, maturation arrest, hypospermatogenesis, and so-called disorganization with sloughing (to me at least a rather confusingdiagnostic pattern).
Sertoli only/Pure Germ Cell Aplasia
In my opinion this is the most obvious pattern associated with infertility. As the name implies, “Sertoli only” tubules show seminiferous tubules populated only by Sertoli cells with no germ cells present. Often affected tubules may also show variable basement membrane thickening (FIG 4). Within a given biopsy, all or a certain subpopulation of tubules may show this pattern. For whatever reason, the tubules seem to remind me of empty cylinders in a revolver. A key point is that this is an irreversible change, no sperm can be salvaged from these tubules as no germ cells are or will ever be present. Using the previous analogy, once the cylinders are empty, the revolver can never again be loaded.
This is an end-stage change, and as such can be seen associated with a number of conditions including adult cryptorchid testis (even if descended when young), so-called idiopathic infertility, chromosomal anomalies, structural abnormalities of the Y-chromosome, hypogonadotrophic hypogonadism, orchitis, post radiation or chemotherapy, in estrogen and anti-androgenic therapy, and as a consequence of chronic hepatopathy.
As implied by the name, maturation arrest is characterized by a block in the maturation to spermatids (therefore no mature sperm or acrosperms are present – refer to hypospermatogenesis for more information). Affected tubules tend to arrest at either the primary spermatocyte stage (round germ cells with an open or clumped tigroid/so-called spireme chromatin pattern) or at the spermatogonia (dark cells morphologically distinct from sertoli cells, which lie at the basement membrane, FIG 5) stage.
Clinically, there are many differential causes of maturation arrest including a history of undescended testes, excessive alcohol (or other toxic agent) consumption, chronic marijuana use, cytotoxic chemotherapy, and hypogonadotrophic hypogonadism. It is important to keep in mind that some cases of maturation arrest are reversible, providing hope for sperm retrieval and fertility.
Hypospermatogenesis is defined as an equivalent decrease in the numbers of spermatogonia and primary spermatocytes. The key difference from tubules showing maturation arrest is the presence of mature spermatids. In hypospermatogenesis all elements arepresent, albeit decreased in number (FIG 6). Of course, it is very easy to make a comment that in hypospermatogenesis spermatocytes and spermatids are decreased, but what is the reference for normal spermatogenesis? How do we quantify hypospermatogenesis? One method involves establishing the germ cell to Sertoli cell ratio (a ratio of 13:1 is considered normal in a healthy young man,approximately 12 Sertoli cells per tubule is considered normal and about half of the germ cell elements should be in the spermatid stage). What we are looking for are mature sperm or acrosperm (mature sperm characterized by small dark nuclei with a focal clear cap) and if necessary, touch or scrape preparations stained with either Diff-Quik or H&E can be used to examine the sperm.
Clinically, hypospermatogenesis can be associated with hormonal dysregulation, congenital germ cell deficiency, Sertoli cell deficiency (without the nurturing mother, the children do not mature well), Leydig cell dysfunction, androgen insensitivity, chemical agent exposure, exposure to heat or radiation, and even varicocele.
So-called disorganization with sloughing
This is yet another pattern sometimes described in infertility biopsies in which spermatogenesis has a disorganized appearance and lumina are filled with sloughed immature cells. Interestingly I could not find a decent color image illustrating this pattern and personally, I generally avoid using this term for various reasons. A pattern similar to this can be seen in both hypospermatogenesis and obstruction. Even rough handling of the biopsy can cause artifacts mimicking this pattern!
Interestingly, active or normal spermatogenesis (FIG 7) can be seen over one fourth of infertility biopsies! A normal biopsy seen tern/Active spermatogene sis. There is abundant full range maturation in all the setting of infertility suggests an obstruction in or absence of some part of tubules present, and infertility may be due to the ductal system. Specific causes of an obstruction. In this obstruction may include varicocele, case the revolver is loaded, but maybe the “barrel is obstruction of the rete testes, angiectasias, blocked.”and Young’s syndrome (obstructive azoospermia with chronic sinopulmonary infections).
Writing the report
Once the biopsy has been reviewed and the various patterns have been identified, I generally report them (including normal), much as one would do in reporting a mixed germ cell tumor.
Additional features that should be mentioned include the degree of peritubular fibrosis and tubular sclerosis (as these represent end-stage changes), the state of the Leydig cells in the biopsy, and a comment regarding the presence or absence of intratubular germ cell neoplasia and/or tumor.
Leydig cell hyperplasia/nodules can be seen in patients with choriocarcinoma, seminoma, nonseminomatous germ cell tumors with syncytiotrophoblast giant cells, hCG treatments, varicocele, androgen insensitivity, Leydig cell dysfunction, five alpha reductase inhibitor therapy, and nonsteroidal anti-androgens.
Intratubular germ cell neoplasia (ITGCN) is characterized by large cells within the tubules with marked cytologic atypism, manifested by prominent nucleoli and cytoplasmic halos (FIG 8). If there is any question regarding the presence of ITGCN, a condition that can be thought of as “seminoma in-situ”. Immunoperoxidase stains for PLAP, Ckit (CD117), Podoplanin (D2-40) and Oct 3/4 (all readily available at ProPath) can be employed to highlight these atypical cells, and separate them from normal immature germ cells. Generally tubules with ITGCN lack spermatogenesis.
Once all these elements have been described the resulting report will provide valuable information which can be correlated with the patient’s clinical and laboratory findings, and provide information guiding possible treatment and fertility techniques. A sample diagnosis would be as follows:
Testis, biopsy submitted for evaluation of infertility (collected 6/1/06):
Testicular parenchyma showing:
— 35% Sertoli only tubules.
— 40% tubules showing hypospermatogenesis.
— 25% normal tubules with active spermatogenesis.
— Few sclerosed tubules are present (2-3 overall).
— No Leydig cell nodules/hyperplasia are noted.
— Negative for intratubular germ cell neoplasia (ITGCN) and invasive tumor.
In conclusion I hope readers find this review helpful and interesting. As always, at ProPath, we are committed to helping our clinical colleagues through providing the best in diagnostic pathology in a timely fashion. Please do not hesitate to contact me if I can be of assistance.
1. Nistal M, Paniagua R. Testicular Biopsy Contemporary Interpretation. Urologic Clinics of North America 1999; 26(3): 1-41. (A very thorough review of both pediatric and adult testicular biopsy)
Special thanks to Dr. Jonathan Epstein for his permission in using images from Epstein JI, Yang XJ. Prostate biopsy interpretation, Philadelphia: Lippincott, Williams & Wilkins, 2002.
Date of last revision: October 2008.