Subfertile Men Research Articles

The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men

We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4±1.3 to 12.5±1.9 million/ml). Treatment significantily reduced ROS (mean±SEM) (775.3±372.2 to 150.3±105.2×103counts/10 second) and 8-OH-dG (45.3±10.4 to 16.8±3.3 fmol/μg DNA). Treatment increased the AR (55.1±2.2 to 71.6±2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55–38.1).

Ubiquitin-based Method for Semen Analysis and Male Factor Infertility-Diagnosis in Men

Objectives: Sperm morphology is somewhat correlated with fertility in men, yet the existing, subjective sperm morphology assays provide only a limited insight into patient’s infertility. We have recently discovered that the defective sperm of humans and other mammals become ubiquitinaned on their surface during epididymal passage and partially resorbed. Ubiquitination is the process in which denaturated or redundant proteins are covalently linked to a small (8.5 kDa) proteolytic marker ubiquitin and consequently recognized by the cellular proteolytic machinery residing in the lysosomes and/or proteasomes. The objective of this study was to establish the relationship between the level of sperm ubiquitination and fertility in the semen of fertile and subfertile men.

Cytokine concentrations in seminal plasma from subfertile men are not indicative of the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract.

The inflammatory response to the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract of subfertile men without any signs or symptoms of infection was investigated by measuring the concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in seminal plasma. Semen samples were collected from 30 culture-positive subfertile males and 23 culture-negative subfertile males. Enzyme-linked immunosorbent assays showed that IL-8 was present in relatively high concentrations (0.12-4.8 ng/ml) in all semen samples investigated. In contrast, the other cytokines were only detectable in 72% (IFN-gamma), 44% (IL-6) and 19% (TNF-gamma) of the samples and were present in relatively low concentrations (1-410 pg/ml). Seminal plasma cytokine concentrations were similar in samples from culture-positive and culture-negative males. These data strongly indicate that the presence of U. urealyticum or M. hominis in the lower genital tract of subfertile males reflects a silent colonisation rather than infection.

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound.

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

ASSOCIATION OF UREAPLASMA UREALYTICUM WITH ABNORMAL REACTIVE OXYGEN SPECIES LEVELS AND ABSENCE OF LEUKOCYTOSPERMIA

Ureaplasma urealyticum is a commensal of the lower genitourinary tract of many sexually active adults. The organism is more common in partners of infertile than fertile marriages. We conducted a prospective study at our tertiary care center to confirm a possible association between U. urealyticum and abnormal sperm function parameters. A total of 50 consecutive male patients seeking general urology consultation for lower urinary tract symptoms characteristic of chronic prostatitis were evaluated. Urine and semen localization cultures were performed with additional semen cultures for U. urealyticum, Chlamydia trachomatis and Mycoplasma hominis. Specimens from 21 healthy men were used as controls. Specimens were analyzed by a computer assisted semen analyzer, and verified manually for concentration, percent motility and morphology. Leukocytospermia was measured by the Endtz test. Semen specimens were also analyzed for reactive oxygen species (ROS), acrosome reaction and mannose binding assay. Of the patients 17 had positive U. urealyticum cultures and the other cultures were negative. Patients with U. urealyticum had significantly higher ROS levels (log [ROS + 1] = 2.52 +/- 0.25) than those without U. urealyticum (1.49 +/- 0.20, p = 0.002) or controls (1.31 +/- 0.19, p = 0.002). Leukocytospermia was detected in only 1 of the 17 (6%) positive specimens and 4 (12%) negative specimens. Seminal ROS levels are elevated among patients with U. urealyticum. ROS induces lipid peroxidation, which reduces membrane fluidity and sperm fertilization capability, and may be the mechanism by which U. urealyticum impairs sperm function. Absence of leukocytospermia does not exclude U. urealyticum.

Effects of long-term treatment with human pure follicle-stimulating hormone on semen parameters and sperm-cell ultrastructure in idiopathic oligoteratoasthenozoospermia.

Ten subfertile men affected by idiopathic oligoteratoasthenozoospermia and exhibiting normal serum hormone levels received a long-term treatment with human pure follicle-stimulating hormone (hp-FSH) (150 IU, intramuscularly, three times per week for 6 months). Semen parameters and ultrastructural features of spermatozoa were evaluated before and after therapy. The results showed an increase in sperm cell concentration and, more interestingly, motility. Electron microscopic examination revealed an improved fine architectural pattern, mainly involving acrosome, head and chromatin and middle-piece, in accordance with the positive changes of functional data. No significant changes of hp-FSH treatment on serum hormone levels were observed, since the latter were found to be substantially unchanged after 6 months of therapy. The present data suggest: (i) the benefit of hp-FSH administration in idiopathic oligoteratoasthenozoospermia, when hormone parameters support a substantial integrity of spermatogenetic microenvironment and (ii) an optimal effect after long-term (6 months) therapy.

Sperm chromatin packaging as an indicator of in-vitro fertilization rates.

The development of a sequential diagnostic schedule for patients consulting for infertility disturbances would be an ideal method of approach for clinicians in the absence of an aetiological or pathophysiological diagnosis. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology as well as in-vitro fertilization (IVF) rates, with chromatin staining among fertile and subfertile men. Two semen smears were prepared from each specimen obtained from 72 men to record normal morphology and chromatin packaging as recorded by chromomycin A(3) (CMA(3)) staining. Following the semen analyses, the 72 men were divided into the two morphological groups, namely <4% and >4% normal forms. Significantly different percentages of CMA(3) staining (mean +/- SE) were recorded between the two morphological groups, namely 65.9% +/- 3.5 and 44.5% +/- 1.7 (P +/- 0.001). A highly negative significant correlation existed between percentage of normal morphology as recorded by strict criteria and CMA(3) staining. A highly significant and positive correlation was recorded for normal morphology and IVF rates (r +/- 0.45, P +/- 0.0001). A significant negative correlation (r +/- -0.51, P +/- 0.0001) existed between CMA(3) values and IVF rates. The discriminating power of nuclear maturity, as recorded by CMA(3) staining, to identify abnormal morphology values and poor IVF rates was calculated with receiver operator characteristic (ROC) analyses. The areas under the ROC curves were 0.86 for sperm morphology and 0. 74 for IVF rates. The calculated threshold values for CMA(3) staining to distinguish between morphology groups were 48 and 50% for IVF. Chromatin packaging assessment is a valuable addition to the sequential diagnostic programme in an assisted reproductive arena.

Optimal use of infertility diagnostic tests and treatments. The ESHRE Capri Workshop Group.

The general definition of infertility is a lesser capacity to conceive than the mean capacity of the general population and infertile couples can be characterized in two groups: those unable to conceive without therapy and those who are hypofertile, but conceive without therapy. The initial diagnostic tests for infertility should include a midluteal phase progesterone assay, a semen analysis and a test for tubal patency such as a hysterosalpingogram. Measuring progesterone is the best test for confirming ovulation. To predict ovulation, evaluating the luteinizing hormone (LH) surge is the best single assay while measurement of LH plus preovulatory oestrogen is the best prediction. Today primary investigation of the morphology of the uterus and tubes should be by hysterosalpingography. However, ultrasound, particularly with simple contrast media, is likely to gain in importance. Laparoscopy should be reserved as a further diagnostic procedure or in combination with endoscopic surgery. There are situations in which semen analysis is of utmost importance and of absolute predictive value, namely, in cases of azoospermia. In general semen analysis remains a substantial part of the fertility workup, but any consideration of its predictive value has to be cautious. Performing genetic tests before, during and after assisted reproductive techniques (ART) is an intrinsic part of good clinical practice. These tests allow one to reach a correct diagnosis, to give adequate genetic counselling to the couple and their families in cases such as (i) women with Turner syndrome; (ii) men with 47, XXY; (iii) men or women with structural chromosomal aberration; (iv) men with Yq11 deletion or (v) men with congenital bilateral absence of vas deferens. Patients should, of course, be made aware of the occurrence of de-novo mutations taking place in the testis and in the embryo. Treatment of some causes of infertility are of proven value. For example induction of ovulation. Others are more controversial. Among the many empirical treatments suggested for the treatment of the various form of subfertility, surgical treatment of varicocele in the male, treatment of pelvic endometriosis in the female and the efficacy of the ART strategies offered to the subfertile couple are considered. Many varicocele studies are of poor quality. A few are good, but small in size. They do not show an improvement in pregnancy rates. Therefore, at the moment, there is insufficient scientific evidence for recommending routinely surgical treatment in subfertile and/or oligozoospermic men with a varicocele. Randomized, double-blind controlled trials demonstrated the modest efficacy of endometriosis ablation in increasing the pregnancy rate in infertile women while drugs suppressing ovulation are of no benefit to infertile women with endometriosis. Although the largest body of evidence available suggests that IVF success declines in repeated ART cycles, an accurate estimate of the true success rate in the 'nth' cycle of IVF treatment is not possible. Similarly little is still known of the reasons for the overall low continuation rates with IVF treatment.

Significance of simultaneous determination of serum and seminal plasma alpha-tocopherol and retinol in infertile men by high-performance liquid chromatography.

High-performance liquid chromatography was used for the simultaneous determination of alpha-tocopherol and retinol in serum and semen of 40 subfertile men. The serum levels of the two vitamins were significantly higher in serum than in semen (3- to 10-fold) (P < 0.001). The mean alpha-tocopherol levels were higher in the serum and semen of men with normal sperm parameters (20 +/- 5 and 5 +/- 4 mumol L-1) than those with oligozoospermia (10 +/- 4 and 3 +/- 2 mumol L-1), azoospermia (8 +/- 3 and 3 +/- 1 mumol L-1) and asthenozoospermia (9 +/- 6 and 3 +/- 2 mumol L-1) (P < 0.002). Mean retinol levels in serum were similar in men with normal sperm parameters (2.4 +/- 2 mumol L-1) as in those with defective sperm parameters such as oligozoospermia (2.5 +/- 2 mumol L-) and asthenozoospermia (2.1 +/- 1.0 mumol L-) (P = 0.15), but significantly lower in men with azoospermia (1.3 +/- 0.3 mumol L-1) (P < 0.05). The alpha-tocopherol:retinol ratio was significantly higher in semen than in serum of men with normal sperm parameters (11.5) and those with asthenozoospermia (10.3) compared with ratios in those with oligozoospermia (8.3) and azoospermia (6.3). This may be related to reduced antioxidant activity in sperm dysfunction as a result of lipid peroxidation, from increased generation of reactive oxygen species.

Flow cytometric analysis of semen preparation, and assessment of acrosome reaction, reactive oxygen species production and leucocyte contamination in subfertile men

Reactive oxygen species (ROS) production, poor acrosome reaction ability, and leucocyte and round cell contamination of semen are associated with diminished male fertility. In addition to the assessment of these variables, the number of abnormal and round cells in semen preparation was studied by flow cytometry. Men with varying degree of fertility despite normal concentrations and motility of spermatozoa were investigated. Group 1 consisted of semen donor candidates (n = 25), group 2 of patients with acceptable fertilization in vitro, but diminished fertility in vivo (n = 56), and group 3 of patients with poor (< 25%) fertilization in vitro (n = 32). The subfertile men in groups 2 and 3 had lower concentrations and poorer rapid progressive motility of spermatozoa than the men in group 1. Using flow cytometry, the number of events in the round cell gate in the Percoll-prepared semen sample was higher in the subfertile men than in the donors, independently of the differences in other semen parameters. However, there were no significant differences between the groups in the number of round cells in native semen by microscopic analysis, or in ROS production or acrosome reaction. In conclusion, flow cytometric assessment of round cells may be a useful method in the investigation of male fertility.

Transurethral deroofing of midline prostatic cyst for subfertile men.

We evaluated the efficacy of transurethral deroofing of a midline prostatic cyst in subfertile men with one or more of the following semen abnormalities: decreased ejaculatory volume, decreased sperm motility and oligo- or azoospermia. Results from treatment of a series of 11 subfertile men with a midline prostatic cyst by transurethral deroofing of the cyst are presented. Five patients showed an improvement of seminal volume. Only one patient demonstrated an improvement of sperm count. Sperm motility was not influenced. No relationship was found between positive outcome following operation and either size of the cyst or dilatation of the seminal vesicles. Spontaneous pregnancies did not occur after transurethral deroofing of the midline prostatic cyst. In conclusion, our study suggests a poor efficacy of transurethral deroofing of a midline prostatic cyst in subfertile men with the above mentioned semen abnormalities.

Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men.

The introduction of intracytoplasmic sperm injection (ICSI) has revolutionized treatment of male-factor infertility. Even with a single spermatozoon a pregnancy can be achieved. In cases of azoospermia due to obstruction or highly impaired spermatogenesis, spermatozoa can be retrieved directly from testicular tissue recovered by testicular biopsy followed by sperm extraction. The predictive value of histology from semi-thin sections of testicular biopsies was assessed in relation to testicular sperm extraction (TESE) results, using 1418 biopsy samples from 766 subfertile men which were evaluated simultaneously using a modified Johnsen score and an ordinal classification system for spermatozoa in TESE samples. In 655 men bilateral samples were available. Based on histological findings and TESE results, the quality of spermatogenesis in the right testes was significantly better than that in the left testes. There was a difference between the two sides in 35.7% of all patients for histology and 32.7% for TESE results. When best results from either testis were used for analysis, 76.9% of all men revealed spermatozoa in TESE preparations, although during histological evaluation of semi-thin sections only 64% of all men had shown mature spermatids. In a core group of 250 azoospermic men without anamnestic hints to obstruction and most likely to benefit from ICSI, TESE was successful in 62.8% men. Subdivision of this group dependent on follicle stimulating hormone (FSH) serum concentrations revealed that even in cases of increased FSH concentration, between 39.1 and 64.7% of men showed mature spermatids in their TESE samples. A subset of 70 azoospermic men from the main sample with symptoms and history suggestive of an obstruction and considered as positive controls showed a positive TESE result in all patients. The histology had failed to predict this in 2.9% of all cases. Nevertheless, in five men an early stage of testicular tumour (carcinoma in situ = CIS) was detected. Two of these males suffered from bilateral CIS. This reflects a prevalence of 0.7% testicular malignancy in the group of patients without a history of excurrent duct obstruction. The data demonstrate that a trial TESE with histology based on the semi-thin sectioning technique is a powerful diagnostic and therapeutic procedure, which justifies the invasive nature of sperm retrieval for ICSI. In addition, the results stress the importance of bilateral biopsies to gain optimal diagnostic and therapeutic results.

Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions.

The contribution of leukocytes and of spermatozoa to reactive oxygen species (ROS) production in prepared sperm suspensions from donors and subfertility patients was compared. In both groups, more leukocytes/10(6) spermatozoa were counted in samples which produced detectable ROS than in those that did not: Donors-645 vs. 170 (medians, n = 7; p < 0.01, Kruskal-Wallis), Subfertile group-1785 (n = 18) vs. 11 (n = 8) (p < 0.005, Kruskal-Wallis), respectively. Leukocyte concentrations were correlated with basal (r = 0.826, p < 0.001) and with ROS production stimulated with 50 mumol N-formyl, met, leu, phe l-1 (N-FMLP) (r = 0.835, p < 0.001) and 100 nmol phorbol 12-myristate 13-acetate l-1 (PMA) (r = 0.835, p < 0.001) measured using a chemiluminescence assay. Leukocytes were removed from the sperm suspensions of 6 donors and from 96 ejaculates from 21 subfertility patients and ROS production was determined. Subsequently, in all 6 donors, N-FMLP did not stimulate ROS production indicating that leukocyte removal was complete, though in one case PMA stimulated low levels of ROS production. In 65 ejaculates from subfertile men the N-FMLP response was completely eliminated but in 7 of these samples PMA continued to stimulate ROS production. We conclude that infiltrating leukocytes are the predominant source of ROS production in unpurified sperm preparations. Some purified sperm suspensions could be stimulated to produce ROS by the addition of PMA indicating that spermatozoa themselves may produce ROS, albeit in much smaller amounts.

Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men.

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.

Are sperm motion parameters influenced by varicocele ligation?

Objective: To document objectively, improvements in sperm motion parameters in subfertile men after varicocele ligation with the use of computer-aided sperm analysis. Design: Retrospective chart review. Setting: An academic clinical environment. Patient(s): Thirty-four men with varicocele as the primary cause of infertility who underwent a physical examination, hormonal profile, and Doppler ultrasound evaluation. Intervention(s): Computer-aided sperm analysis was performed before and after either subinguinal or laparoscopic varicocele ligation. Main Outcome Measure(s): Patients were followed up for 3–18 months after operation, and average computer-aided sperm analysis values were obtained before and after operation. Computer-aided sperm analysis values were analyzed by paired Student’s t-test, with special reference to motility and sperm motion parameters. Result(s): After varicocele ligation, patients demonstrated increases in mean sperm density and in the overall distribution of sperm with rapid velocity, although the increases were not statistically significant. Mean measures of straightness, linearity, and track speed also were increased after operation, but only progressive sperm velocity was increased to a statistically significant level. Conclusion(s): Because progressive velocity has been a primary computer-aided sperm analysis parameter predictive of male fertility and the success of assisted reproductive techniques, we conclude that varicocele ligation results in improvements in semen parameters, especially the key parameter of progressive sperm velocity.

Imaging of the Male Reproductive Tract

Radiologic imaging may often play a critical role in the evaluation and management of patients with male factor infertility. Historically, the role of radiologic imaging was confined to vasography in the diagnosis of obstructive azoospermia. Today, radiologic imaging modalities have three indications in subfertile men. The first is the evaluation and management of obstruction of the excurrent ductal system in patients with azoospermia. Vasography, transrectal ultrasonography (TRUS), or seminal vesiculography is performed in this situation. The second indication is the evaluation and management of varicoceles, for which either venography or scrotal ultrasonography is routinely performed. Finally, radiologic imaging studies of the upper urinary tract are indicated for patients with congenital anomalies of the Wolffian duct system. Ultrasonography is the modality of choice for screening the upper tracts of men with vasal agenesis. Patients with bilateral vasal agenesis have a much lower frequency (< 20%) of upper tract abnormalities than patients with unilateral vasal agenesis (up to 70%). This chapter specifically discusses imaging modalities used in the management of obstruction and varicoceles.

Oral selenium supplementation increases sperm motility in subfertile men

Oral selenium supplementation increases sperm motility in subfertile men

Risk of testicular cancer in subfertile men: case-control study

Objective: To evaluate the association between subfertility in men and the subsequent risk of testicular cancer.Design: Population based case-control study.Setting: The Danish population.Participants: Cases were identified in the Danish Cancer...

Subfertile men with constitutive chromosome abnormalities do not necessarily refrain from intracytoplasmic sperm injection treatment: a follow-up study on 75 Dutch patients.

A follow-up study was performed to investigate the impact of the detection of a chromosome abnormality in infertile men who are candidates for intracytoplasmic sperm injection (ICSI) treatment. In this collaborative study between clinical genetics centres and fertility clinics in the Netherlands, 75 ICSI couples of which the male partners had a chromosome abnormality were included. All couples were extensively counselled on the risk of having a chromosomally unbalanced child. Forty-two out of 75 couples chose to proceed with the ICSI treatment. So far, treatment has resulted in a pregnancy in 11 cases. Four of them opted to have invasive prenatal diagnosis. Despite the genetic risks related to a chromosome abnormality in infertile men, a small majority (56%) of the couples did not refrain from the ICSI treatment.

Ultrastructure of peritubular tissue in association with tubular hyalinization in human testis

Testicular peritubular tissue, also known as the tunica propria, surrounds the seminiferous tubules and is responsible for contractile, paracrine and transport functions. The aim of the present report is to describe the pathomorphology of peritubular tissue in association with tubular hyalinization in human testis. Twenty-seven testicular biopsies from 21 subfertile and infertile men were studied with the electron microscope. Biopsies from five patients showed complete or nearly complete tubular hyalinization. In addition to changes described earlier, the following new ultrastructural features were observed: 1. loss of polarity and configuration of myoid cells; 2. protrusion of myoid cells towards the tubule and evagination of basal lamina surrounding the tubule towards the interstitial direction leading to ‘bridge’ formation. These ‘bridges’ of myoid cells often created completely separated small compartments within the tunica propria; 3. vacuolization and fragmentation of myoid cell nuclei; 4. a balloon-like swelling of myoid cell containing phagolysosomes and lipid droplets.We conclude that disorganization and loss of vital functions of the extracellular matrix and myoid cells contribute to the pathogenesis of tubular hyalinization.