Semen, sperm anomalies and infertility - Reproductive Pathology

Atlas of Clinical Andrology

Chapter 6. Semen, sperm anomalies and infertility

Sperm are antigenic when injected subcutaneously into females. Thus, there are several physiological mechanisms to remove sperm from the female reproductive tract after intercourse. This removal takes the form of phagocytosis; deposition of sperm induces an influx of phagocytic cells (neutrophils, macrophages and dendritic-like cells) into the reproductive tract. Seminal plasma contains various molecules that can inhibit lymphocyte activation. Lymphocytes in the lymph nodes draining the uterus can become activated by seminal plasma, perhaps because immuno- suppresants are inactivated as sperm antigens reach the lymph nodes. Despite the presence of mechanisms to prevent immune responses to sperm, females can develop anti-sperm immunity, and antibodies directed towards sperm can occasionally be recovered from the reproductive tract (Hansen, 2000).

In oligozoospermic men there is a higher rate of apoptotic DNA in sperm. In fertile men, the simple selection of motile sperm can eliminate most of the apoptotic sperm. However, the higher percentage of apoptotic sperm from severe oligoasthenozoospermic men could lead to the risk of injection of sperm with fragmented DNA.

SEMINAL PLASMA

Male accessory sex organs, made of epithelial/ mesenchymal components, require androgens for proliferation and maintenance of their function. There are three types of epithelial cells: secretory glandular cells, non-secretory basal cells and neuroendocrine cells:

(1) Basal cells, which are stem cells for secretory epithelial cells, are androgen-independent because they do not express androgen receptors.

Table 6.1 Some biochemical markers to identify the source of secretions within the ejaculate by the presence of different ejaculate components

Source of secretion	Biochemical markers
Testes	Androgen-binding protein Inhibin Testosterone Transferrin
Epididymal ducts	Carnitine Glycerophosphorylcholine Inositol
Seminal vesicles	Fructose Prostaglandins
Prostate	Acid phosphatase Citrate Calcium, zinc Spermine Vesiculase
Bulbourethral/urethral glands	IgA Mucoproteins

(2) Neuroendocrine cells play a role in regulating the growth and function of secretory cells.

(3) Mesenchyme comprises smooth muscle cells, fibroblasts, lymphocytes and neuromuscular tissue embedded in an extracellular matrix.

Several biochemical markers are used to identify the source of secretions within the ejaculate by the presence of different components (Table 6.1).

Figure 6.1 (a) Sperm membrane regions. Reproduced with permission of Hemisphere Publishing. (b) Plasma membrane surface domains: (1) the acrosomal segment with some particles; (2) the equatorial segment showing a striated morphology; (3) the post-acrosomal segment with abundant particles; and (4) the tail segment (middle piece and main piece) with dispersed particles. (From Calamera (1998), with permission)

SPERM ANOMALIES

The World Health Organization (1999) has developed ‘strict criteria’ for sperm normality. Several classifications have been designed to describe sperm abnormalities (Figures 6.1-6.5; Tables 6.2 and 6.3). The following groups are of special physiological significance:

(1) Head abnormalities (Figures 6.6-6.9):

(a) large heads;

(b) small heads;

(d) duplicated heads;

(e) amorphous heads;

(f) loose heads;

(2) Tail abnormalities;

(3) Neck/middle piece abnormalities;

(4) Cytoplasmic droplets.

Every man has a certain percentage of sperm that are defective in one or more attributes. Such sperm are unlikely to be able to fertilize an oocyte. Many sperm are defective in more than one attribute. Also, many sperm that are not detected as defective may have a problem that prevents them from fertilizing an oocyte. There are several laboratory assays which can predict if an individual male is likely to be subfertile or sterile. Subfertile men may be able to fertilize an oocyte by the application of in vitro fertilization (IVF) or intrauterine insemination (IUI). Cleavage is probably the best measure for fertility in IVF, but passage through two to three cleavage divisions is not predictive that an embryo will complete embryogenesis and fetal development. The number of sperm in an IVF droplet or IUI dose is substantially in excess of the minimum number needed to maximize ‘success’, although it is balanced with the requirement to minimize polyspermy.

Table 6.4 summarizes the andrological terminology used to describe semen deviations. Since it is often the practice to describe all deviations from normal semen variables with words, the international nomenclature was introduced, because these terms may have different values in different laboratories.

Sperm surface in HIV patients

In the initial stages of HIV infection, the patient may ejaculate motile (and fertile) sperm. Some patients undergoing highly active antiretroviral therapy (HAART) produce active sperm with normal motility (Figure 6.10). Antiviral regimens affect lymphocytes and sperm chromosomes, morphology and semen quality in HIV patients. HIV particles are present on the sperm membrane as determined by labeling with monoclonal or polyclonal anti-HIV antibodies.

Atomic force microscopy (AFM) is a powerful technique to evaluate any alterations in the sperm of HIV patients, and the effects of HAART including minute details, e.g. viral particles on sperm membrane. AFM, unlike electron microscopy, permits virions to be imaged in almost their natural environment and identification of possible membrane penetration or any budding (Joshi et al., 2000, 2001a, 2001b). An AFM unit (Surface Imaging System ultraobjective; Surface Imaging System, Germany) attached to a conventional Olympus microscope (BX60, Japan) is employed for investigation. Cantilevers of silicon of length 450 μm, width 50 μm and thickness 2 μm are attached to the objective. The spring constant of the cantilever is 15 N/m, operated at frequency 159 266 kHz.

PATHOLOGY OF INFERTILITY

Infertile patients can be classified as having the following conditions: subfertile semen (Figure 6.11), periodic oligozoospermia, permanent oligozoospermia, oligozoospermia, azoospermia (Figure 6.12), hypogonadotropism (pre- and postpuberty) and immunological infertility. The step-by-step scheme for the evaluation of cervical mucus in infertility patients is shown in Figure 6.13.

Figure 6.3 Morphological anomalies of spermatozoa (all are of immotile type, being devoid of mitochondrial thickening in the middle piece). (a) Mitochondria thickening is lacking (arrow).This deformed spermatozoa, being deprived of its ‘motor’, would have been immotile in the ejaculate (12 000x). (b) The constricted boundary of the middle and main pieces of the tail probably represents a mechanically weak point as shown by a spermatozoon broken at this site. The main piece abruptly joins the short end-piece (arrow) (12 000x). (c)-(e) Abnormal spermatozoa from patients with suspected male infertility. (c) Spermatozoon with a monstrous head and a short and irregularly thick tail (10 000 x). (d) Spermatozoon with a rudimentary and bipartite head and two tails (8700 x).(e) Sperm with unusually small, oval and rough-surfaced head. without an acrosome (20 000x). (f) Spermatozoon with two heads found in the same normal semen. The acrosome is probably failing in these heads. Although deformities are found in no more than 15% of the total spermatozoa in normal semen, they occur more or less in excess or even exclusively in the ejaculates of sterile males (10 000)

Non-obstructive azoospermia (NOA)

Once a single viable sperm is injected into a normal metaphase II oocyte it has the potential to achieve successful fertilization, and normal embryo development can proceed. The task of extracting a single viable sperm from an azoospermic man is a specialty in itself. This task is even more complicated in a man with nonobstructive azoospermia (NOA). The diagnosis of azoospermia should include: complete semen analysis, careful inspection of semen sediment obtained by ultracentrifugation of seminal plasma, and clinical examination for the presence of vasa deferentia. The differentiation between obstructive azoospermia and NOA is achieved by testicular biopsy and endocrine profile. The degree of spermatogenesis impairment should then be evaluated. Tesarik et al. (2000) incubated testicular tissues in hormone- and growth factor-supplemented media in vitro in order to stimulate maturation of spermatogenic cells. Hypospermatogenesis provides good chances for isolating viable, intact spermatozoa with fertilizing capacity. Tiny focal spermatogenesis may take place in the testis without sperm appearing in the selected biopsy specimen or released in the ejaculate (Tournaye et al., 1996). In such cases it is possible to isolate sperm from additional testicular specimens and to use them in IVF-intracytoplasmic sperm injection (ICSI) (Ben-Yousef et al., 1999). Careful evaluation of the histology (cytology of the specimen) is crucial to determine the number, location and size of biopsies to be obtained (Ezeh et al., 1998).

Figure 6.5 (a) Semen evaluation using stained semen smear under phase-contrast microscopy. (b) Automatic shaker for proper mixing of semen in calibrated pipettes

Modern diagnostic tools have been used in the management of NOA. Biopsy techniques utilize isolated testicular tissue or spermatogenic cells obtained by testicular sperm extraction (TESE), (open biopsy under local anesthesia) or testicular sperm aspiration (TESA) (fine-needle biopsy) (Craft et al., 1997).

A multiple TESE is superior in cases of NOA because it may enhance diagnostic accuracy and increase the chance of sperm cell retrieval. The sperm isolated from the testis may occasionally have morphology as good as that of ejaculated sperm. The rate of bivalent formation of homologous chromosomes in spermatocytes increases the prospect of focal spermatogenesis in the testes of men with NOA. Bivalent formation can be analyzed with fluorescence in situ hybridization (FISH) using alphasatellite centromere probes for the evaluation of autosomes (Hauser et al., 1998; Paz et al., 2003). Men with NOA have the highest risk for Y-chromosome microdeletions, which are associated with impairment of spermatogenesis. In cases of spermatogenesis or meiotic arrest in men with NOA, techniques are attempted to advance the maturation of spermato- genic cells in vitro. Tesarik et al. (2000) incubated testicular tissues in hormone-supplemented medium in vitro to stimulate maturation of spermatogenic cells.

Leukospermia

Routine application of antibiotics for leukospermia may have a negative impact on semen parameters as well as increasing the possibility of inoculating the reproductive tract of the female partner with antibiotic-resistant bacteria.

The presence or absence of bacteriospermia or leukospermia do not correlate with each other. Leukospermia does not correlate with semen parameters. In a study by Rodin et al. (2003) staphylococcus species were the most common isolates and did not correlate with semen parameters or leukosper- mia. Streptococcus viridians and Enterococcus faecalis were the next most common isolates and were associated with statistically significantly poorer semen quality (Rodin et al., 2003). Leukospermia is a poor marker for either bacteriospermia or impaired semen quality. Staphylococcus species are commonly isolated but appear to be innocuous. Streptococcus viridi- ans and Enterococcus faecalis are associated with poorer semen quality and may warrant treatment.

Azoospermia and oligozoospermia

Azoospermia and oligozoospermia are associated with a wide variety of genetic anomalies. Chromosomal anomalies are more frequent in infertile men compared with fertile men, with a frequency of 2% in all men attending infertility clinics. In azoospermia, the frequency rises to 15%, including about 13% with a 47,XXY chromosome constitution. The remaining azoospermic cases are characterized by specific anomalies, which include other numerical structural anomalies, such as reciprocal and Robertsonian translocations, inversions, ring chromosomes, aneuploidies and supernumerary chromosomes. Congential bilateral absence of the vas deferens (CBAVD) leading to obstructive azoospermia in healthy males is a relatively frequent cause of male infertility, occurring at the rate of 1-2% of cases with sterility and > 6% of cases with obstructive azoospermia. Most men with CBAVD have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, with an increased risk of carrying the 5T variant.

Figure 6.7 Different types of ultrastructural sperm abnormalities as shown by scanning electron microscopy (SEM). Arrows indicate early stage of phagocytosis. Courtesy of Professors Zaneveld and Polakaski, Chicago, IL, USA

Idiopathic oligozoospermia

Several drugs have been used in the treatment of male infertility to improve spermatogenesis or epididymal maturation. However, there is no ‘standard’ therapy for idiopathic oligozoospermia. Various substances, such as kallikrein, vitamins and Kampo (Japanese herbal medicine), have been administered orally as an empiric therapy for male infertility, but the exact mechanism of efficacy has not been established. A novel treatment of male infertility focuses on the role of mast cell blockers, which inhibit the release of histamine and other vasoactive substances from mast cells. The rationale of this approach is based on mastocytosis of the testis in infertile men.

Figure 6.9 Classification of abnormal sperm according to head defects (a)-(f), neck/middle piece defects (g)-(j),tail defects (k)-(m) and cytoplasmic droplet defects (n) as used to evaluate the functional three-dimensional ultrastructure of affected sperm

Table 6.4 Descriptive terminology for semen variables according to WHO guidelines (WHO, 1999)

Term	Definition
Normozoospermia Globozoospermia Oligozoospermia Asthenozoospermia Teratozoospermia Oligoasthenoteratozoospermia (OAT)	Normal ejaculate as defined in Table 6.3 Round-headed acrosomeless spermatozoa < 20 million spermatozoa/ml < 50% sperm with forward progression < 30% spermatozoa with normal morphology Signifies disturbance of all three variables (combinations of only two prefixes may also be used)
Azoospermia Necrozoospermia Hemospermia (hematospermia)	No spermatozoa in the ejaculate All spermatozoa are dead as judged by supravital staining Presence of fresh or altered blood in ejaculate derived from pathology in accessory sexual glands, urethra or bladder. Related to emission and ejaculation; associated with infertility, hematuria, lower urinary tract infection, vascular abnormalities, ductal obstruction or cysts, neoplasms and systemic or iatrogenic factors
Parvisemia	Ejaculate volume < 2 ml
Leukocytospermia -zoospermia -spermia -aspermia	Leukocytes present in ejaculate Refers to sperm in ejaculate Refers to the ejaculate No ejaculate

Several factors could potentially be involved in male unexplained infertility including cytokine-mediated immune response and sperm-membrane lipid peroxidation. Reactive oxygen species (ROS) produced by sperm and white blood cells can attack the sperm membrane, thereby increasing the level of lipid peroxidation. Human sperm are particularly susceptible to oxidative attack, not only because of the high concentration of unsaturated fatty acids within their plasma membrane, but also because of the low concentration of scavenging enzymes in their cytoplasm. The occurrence of any imbalance between ROS production and total antioxidant capacity correlates with male infertility. An increased production of cytokines can increase ROS production in the male genital tract. Male infertility is correlated with increased levels of cytokines in human semen. Urogenital infections increase the concentration of semen cytokines as well as affecting sperm parameters. Studies with pancreatic islet beta-cells have demonstrated a toxic effect of cytokines, mediated by generation of ROS and lipid peroxidation. In vitro studies have demonstrated stimulation of sperm-membrane peroxidation in the sperm of fertile donors, by inter- leukin-a (ILa) and tumor necrosis factor-а (TNF-a).

Figure 6.10 Scanning electron microscopy (SEM) of sperm of normal (a), HIV-infected without treatment (b) and HIV-infected patients treated with highly active antiretroviral therapy (HAART) (c). Higher magnification shows HIV-infected sperm (d) and normal sperm (e). The infected sperm (d) shows complete absence of the central canal, the neck piece is not well marked or is obscured by the sudden increase in width near the neck. From Barboza et al., 2004, with permission

Figure 6.11 Classification of subfertility, periodic and permanent oligozoospermia and azoospermia

Figure 6.12 Flowchart of treatment for azoospermia. CBAVD, congenital bilateral absence of vas deferens; CF, cystic fibrosis; ICSI, intracytoplasmic sperm injection; VV, vasovasostomy; GnRH,gonadotropin releasing hormone; VE, vasoepididymostomy; SV, seminal vesicle; EDO, ejaculatory duct obstruction;TRUS,transrectal ultrasound;TURED, transurethral resection of ejaculatory ducts;TID, therapeutic insemination with donor sperm; TESE, testicular sperm extraction. From Kolettis, 2002, with permission

Figure 6.13 Step-by-step scheme for the evaluation of cervical factor in infertility patients. CM, cervical mucus

Hypogonadotropism

Hypogonadotropism may be acquired either before or after puberty (Table 6.5).

THERAPEUTIC APPROACHES

TO MALE INFERTILITY

Several hormonal and surgical procedures have been used to treat the following major causes of infertility: azoospermia, transurethral resection of ejaculatory ducts (TURED), antegrade ejaculatory duct canalization/balloon dilatation and vasovasostomy microsurgery (Table 6.6).

Azoospermia due to defects in the hypothalamic- pituitary-gonadal axis associated with low hormone levels is treatable. Defects may also be associated with hyposmia or anosmia in patients with Kallmann’s syndrome, hyperprolactinemia hypogonadism with erectile dysfunction/infertility. Nonobstructive azoospermia is treated exclusively by hypogonadal hormones to reinitiate spermatogenesis.

Vasal occlusion can be due to a variety of factors, including previous vasectomy, inflammation and damage as a result of surgery (such as hernia repair) or accident (trauma). Both vasectomy and non-vasectomy patients should undergo a full medical examination and evaluation prior to undergoing vasovasostomy. As with infertility patients, routine assessment should include semen analysis, serum hormone levels and radiographic examination.

Various micromanipulation techniques are used to treat infertility (Table 6.7).

Chromosomal abnormalities/ polymorphisms

Abnormalities in either sex or autosomal chromosomes are frequent in infertile men; therefore, disorders in spermatogenic processes could be directly related to changes in the number or structure of chromosomes. Aneuploidy has been shown in the sex chromosomes of some sterile men. There is an association between alterations in gamete production and genetic disorders, such as Klinefelter’s syndrome or 45,X/46,XY gonadal dysgenesia. This may be due to the presence of the AZF gene (azoospermia factor) on the long arm of the Y chromosome, since deletion of the distal portion of that chromosomal region occurs in azoospermic patients. Other structural abnormalities in sex chromosomes, e.g. microdeletions, isochromosomes, translocations and ring chromosomes, have been found. Also, autosomal alterations, mainly Robertsonian translocations, have been shown in infertile males. Cytogenetic analysis of sterile males is needed not only for the diagnosis of sterility, but also as selection criteria for assisted reproduction techniques, e.g. ICSI.

Table 6.6 Hormonal and surgical procedures for major infertility cases (data from Acosta and Kruger, 1996; Hafez et al., 2003; Hafez and Hafez, 2000;Jeyandran, 2003)

	Treatment
Azoospermia	1500-3000 IU of hCG intramuscularly 3 times a week; recombinant FSH in conjunction with hCG for 6-9 months to initiate spermatogenesis and stimulate Sertoli cells
Transurethral resection of ejaculatory ducts (TURED)	Spinal or general anesthesia plus prophylactic antibiotics; patient prepared in sterile fashion in dorsal lithotomic position; O’Conor sheath used to facilitate digital transrectal prostate massage/manipulation during procedure; standard cystoscopy to inspect urethra/bladder; cystoscope removed to insert electroscope with a resection loop into urethra; same instruments used as for transurethral resection of prostate (TURP); electrical current at 100 W (set for pure cutting); guide wire passed into seminal vesicle with advancement into ejaculatory duct
Antegrade ejaculation duct canalization/balloon dilatation	Cystoscopy used to gain control of distal end of guide wire; Seldinger technique to place 4-mm wide, 2-cm long balloon dilating catheter over guide wire; dilating catheter advanced over guide wire to ejaculatory level duct; proper position confirmed fluoroscopically/visually with cystoscope; dilating balloon inflated/left in place for 2-3 min/balloon inflated; instruments withdrawn to address other side similarly as required; the following complications could be minimized or avoided by careful identification of pertinent anatomical landmark before resection: bleeding, rectal injury, prostate capsule perforation, external sphincter injury with urinary incontinence, bladder neck resection (leading to retrograde ejaculation or a bladder neck contracture)
Vasovasostomy microsurgery	Local or general anesthesia before bilateral, two-layered anastomosis; slight patient movement may cause injury to vas deferens and anastomosis site; pressure points of elbows, hands, ankles, and heels of feet are padded; TED Hose compression boots may diminish risk of deep vein thrombosis; patient offered concurrent sperm retrieval; epididymovasostomy performed if vasovasostomy does not cause patency

hCG, human chorionic gonadotropin; FSH, follicle stimulating hormone

Figure 6.14 Progression of events associated with imbalanced ATP synthesis/degradation and subsequent death of spermatozoa

Molecular parameters of infertility

Severe defects in sperm production cause infertility in 2% of infertile men. Genetic infertility is due to chromosomal abnormalities and microdeletions of the Y chromosome. In azoospermic and oligozoospermic men there are three non-overlapping AZF regions, designated AZFs a, b and c, residing in intervals 5 and 6 of the human Y chromosome. Each of the AZF regions contains many genes that have not been fully characterized. These genes may be potential candidates for AZF. The VCY2 (variable charge, Y chromosome, 2; alias BPY2) gene is located in the most frequently deleted AZFc region in infertile men. Tse et al. (2003) performed testicular biopsy in infertile men with different testicular histologies, then carried out immunohistochemical analysis to determine the possible relationship of VCY2 and the pathogenesis of male infertility. VCY2 was weakly expressed at the spermatogonia and immunonegative in spermatocytes and round spermatids in testicular biopsy specimens, with maturation arrest or hypospermatogenesis. The impaired expression of VCY2 in infertile men suggests its involvement in the pathogenesis of male infertility.

The death of spermatozoa occurs as a consequence of molecular changes in the plasma membrane, cytoplasm and mitochondrial membrane (Figure 6.14).

Immunological infertility

Antisperm antibodies, especially sperm-immobilizing antibodies found in the sera of immunologically infertile women, cause infertility by interfering with fertilization and by blocking penetration through the cervical mucus. There is an immunoglobulin-binding protein capable of binding antisperm antibodies which trap any penetrating sperm. One of the mechanisms by which sperm-immobilizing antibodies exert their anti-fertility effect is by inhibition of the acrosome reaction (AR). Spontaneous AR is almost completely inhibited by sperm-immobilizing antibodies; this inhibitory effect on AR can be abolished by incubation of antisperm antibody-treated sperm in antibody-free medium, as demonstrated in an in vitro human fertilization test, in which the blocking effect of the antibody on the sperm-zona pellucida interaction is reversed by incubation of the antibody-bound spermatozoa in antibody-free medium. Only capacitated sperm undergo AR.

Table 6.8 Endocrine profile of the blood of infertile men

Syndrome	FSH	LH	Testosterone	Total estrogen
Primary hypogonadism	Very little	Very high	Normal or low	Low
Hypogonadotropic hypogonadism	Undetected	Undetected	Low	Very low
Idiopathic oligospermia (maturation arrest at spermatocyte stage)	Normal	Normal	Low	High
Varicocele (left)	Normal	Normal	Normal	Normal
Oligozoospermia (with non-bacterial chronic functional prostatitis)	Very low	Very high	Very low	Very high
Sertoli cell-only syndrome	High	Normal	Normal	Normal

Table 6.9 Endocrine profile and diagnosis of azoospermia