Benjamin Bartoov

In Vivo and In Vitro Impairment of Human and Ram Sperm Nuclear Chromatin Integrity by Sexually Transmitted Ureaplasma urealyticum Infection

Abstract The incidence of Ureaplasma urealyticum infection in the semen of infertile men is variable (7%–42%). Evidence has accumulated through routine semen analysis to suggest that this infection can cause embryo loss without necessarily affecting sperm quality. The aim of this study was to specifically investigate the effects of U. urealyticum infection on sperm chromatin stability and DNA integrity, which are known to be correlated to pregnancy outcome. Sperm cells isolated from human semen infected in vivo with U. urealyticum exhibited a low percentage of stable chromatin as determined by nuclear chromatin decondensation assay (42% ± 4.8%, n = 8) and a high percent of denatured DNA as determined by sperm chromatin structure assay (60.9% ± 9.1%, n = 7). After doxycyclin treatment, a significant improvement in both parameters was observed (73.7% ± 3.6%, P < 0.001 and 30.1% ± 3.5%, P < 0.008, respectively). Sperm cells infected in vitro exhibited higher rates of viability and motility than uninfected cells. In contradistinction, U. urealyticum caused significant dose- and time-dependent chromatin decondensation and DNA damage. The percentage of human sperm cells with denatured DNA increased significantly by 54.9% ± 23.9% and 47.9% ± 12.1%, after 30 min infection with serotypes 8 and 3, respectively, at a multiplicity of infection of 100 ureaplasmas per sperm compared with uninfected control cells. The damage to DNA was significantly more pronounced in infected ram sperm (180.9% ± 21.5%). These results indicate that preserved sperm activity post U. urealyticum infection resulted in damage to paternal DNA, although a high fertilization rate was maintained, and embryonic development may, therefore, be impaired.

Reduced semen quality caused by chronic abacterial prostatitis: an enigma or reality? *

OBJECTIVE To investigate whether there is an inter-relationship between chronic abacterial prostatitis and potential infertility. DESIGN As part of the eligibility studies for hyperthermia treatment for chronic abacterial prostatitis patients, a large number of chronic prostatitis patients were referred to us from peripheral outpatient clinics. Sperm analysis was a routine portion of the eligibility studies. To exclude bacterial prostatitis, urine cultures, expressed prostatic secretion, and semen cultures were performed. The patient population was not differentiated on the basis of those suffering from either nonbacterial prostatitis or prostatodynia according to the commonly accepted classification. The control group was the laboratory normal standard group. SETTING Normal human volunteers in an academic and clinical research environment. PATIENTS The first group includes 86 patients suffering from long-standing (1 to 20 years) chronic abacterial prostatitis, according to the commonly accepted classification, with a mean age of 39.9 +/- 9.5 years. The second group includes 101 normal fertile men with a mean age of 31.4 +/- 5.5 years. INTERVENTIONS The routine semen analysis performed included biochemical tests of seminal plasma, bacteriology, and light microscopy. MAIN OUTCOME MEASURE The original hypothesis was based on a reduction in semen quality in these patients caused by chronic abacterial prostatitis. Measurements for sperm motility parameters, morphology characteristics, prostate markers, and white blood cells (WBC) were designed accordingly. RESULTS Statistical comparisons of the two groups showed that several sperm motility parameters, morphology characteristics, prostate markers, and WBC are outside of the normal value ranges in the chronic abacterial prostatitis group. In addition, there is a correlation between the duration of the disease and two important sperm analysis variables: increased prostatic markers and appearance of sperm morphological defects. CONCLUSION From the results obtained, the high incidence of secondary infertility in these patients may be explained.

Morphological Characterization of Abnormal Human Spermatozoa Using Transmission Electron Microscopy

A morphological analysis of ejaculated human abnormal spermatozoa is presented. Eighteen different malformations are characterized, based on (a) the cells organelle where they appeared, (b) the possible developmental stage when they occurred, and (c) the possible mechanism responsible for a specific appearance. The forces that govern the shaping of the spermatozoan head are described in detail. The possible mechanism responsible for chromatin subcondensation, acrosome malformation, kinked tail forms, and mitochondrial malformations are discussed.

EFFECT OF ACUPUNCTURE ON SPERM PARAMETERS OF MALES SUFFERING FROM SUBFERTILITY RELATED TO LOW SPERM QUALITY

The aim of this prospective controlled study was to assess the effect of acupuncture on the sperm quality of males suffering from subfertility related to sperm impairment. Semen samples of 16 acupuncture-treated subfertile patients were analyzed before and 1 month after treatment (twice a week for 5 weeks). In parallel, semen samples of 16 control untreated subfertile males were examined. Two specimens were taken from the control group at an interval of 2-8 months. The expanded semen analysis included routine and ultramorphological observations. The fertility index increased significantly (p < or = .05) following improvement in total functional sperm fraction, percentage of viability, total motile spermatozoa per ejaculate, and integrity of the axonema (p < or = .05), which occurred upon treatment. The intactness of axonema and sperm motility were highly correlated (corr. = .50, p < or = .05). Thus, patients exhibiting a low fertility potential due to reduced sperm activity may benefit from acupuncture treatment.

ART success and in vivo sperm cell selection depend on the ultramorphological status of spermatozoa

Summary. Management of male infertility has recently shifted from treatment of the subfertile man towards techniques of assisted reproduction (ART). This study aimed to evaluate the possible role of the ultramorphological status of the spermatozoon with respect to sperm selection in vivo and prediction of ART success. Ultramorphological sperm parameters were assessed retrospectively for 92 males with sufficient sperm density (107 spermatozoa ejaculate−1) whose wives conceived following a stepwise discarding of the female genital tract barriers, using intra‐uterine insemination (IUI) (n= 26), in vitro fertilization (IVF) (n= 45) or intracytoplasmic sperm injection (ICSI) (n= 21). In parallel, sperm samples of 71 fertile males were examined. Normal ultramorphology of all head and tail subcellular organelles was found to be essential for the ability of spermatozoa to pass the lower female genital tract. The ultramorphological migration threshold for this barrier is apparently higher than that essential for oocyte fertilization. No specific indication associated with passage through the upper genital tract was found. A high prevalence of axonema defects was found to impair the ability of sperm cells to penetrate the oocyte investment. The natural fertility index, based on routine sperm parameters and the ultrastructural status of the spermatozoons subcellular organelles was confirmed to be beneficial for directing patients to ART. A discriminative score based on axonema integrity was found to contribute additional information for the first choice decision between conventional ART and ICSI (75% prediction ability). Thus it may be helpful in finding the simplest and least expensive procedure with the greatest long‐term chance for pregnancy.

Does acupuncture treatment affect sperm density in males with very low sperm count? A pilot study

Summary. Classic therapies are usually ineffective in the treatment of patients with very poor sperm density. The aim of this study was to determine the effect of acupuncture on these males. Semen samples of 20 patients with a history of azoospermia were examined by light microscope (LM) and scanning electron microscope (SEM), with which a microsearch for spermatozoa was carried out. These examinations were performed before and 1 month after acupuncture treatment and revealed that the study group originally contained three severely oligoteratoastheno‐zoospermic (OTA), two pseudoazoospermic and 15 azoospermic patients. The control group was comprised of 20 untreated males who underwent two semen examinations within a period of 2–4 months and had initial andrological profiles similar to those of the experimental group. No changes in any of the parameters examined were observed in the control group. There was a marked but not significant improvement in the sperm counts of severely OTA males following acupuncture treatment (average = 0.7 ± 1.1 times 106 spermatozoa per ejaculate before treatment vs. 4.3 ± 3.2 times 106 spermatozoa per ejaculate after treatment). A definite increase in sperm count was detected in the ejaculates of 10 (67%) of the 15 azoospermic patients. Seven of these males exhibited post‐treatment spermatozoa that were detected even by LM. The sperm production of these seven males increased significantly, from 0 to an average of 1.5 ± 2.4 times 106 spermatozoa per ejaculate (Z = ‐2.8, P≤0.01). Males with genital tract inflammation exhibited the most remarkable improvement in sperm density (on average from 0.3 ± 0.6 times 106 spermatozoa per ejaculate to 3.3 ± 3.2 times 106 spermatozoa per ejaculate; Z = ‐2.4, P≤0.02). Two pregnancies were achieved by the IVF‐ICSI procedure. It is concluded that acupuncture may be a useful, nontraumatic treatment for males with very poor sperm density, especially those with a history of genital tract inflammation.

Protective effect of the immunomodulator AS101 against cyclophosphamide-induced testicular damage in mice

BACKGROUND Cyclophosphamide (Cy), a widely used anticancer drug, is associated with significant testicular damage and sterility. Co-administration of the immunomodulating compound AS101 during chemotherapy treatments was previously shown to protect organs against cytotoxic damage, without attenuating the drugs anticancer effect. In this animal study, we investigated the effect of AS101 on testicular damage, sperm DNA damage and infertility induced by Cy. Akt and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation were investigated as a possible chemoprotective mechanism. METHODS Mature male mice, 10 in each group, were injected intraperitoneally with 200 mg/kg Cy once a week for 5 weeks, with or without concurrent treatment with 10 microg per mouse AS101 three times per week. Damage to testicular tubules and sperm production was determined, sperm chromatin damage was analyzed and fertility was gauged. Akt and GSK-3beta phosphorylation were evaluated. RESULTS Co-treatment with AS101 during the course of Cy administration significantly reduced the percentage of damaged seminiferous tubules (76.0 +/- 10.8% versus 40.3 +/- 2.6%), and reduced sperm DNA fragmentation (%DFI) from 44.7 +/- 1.0% to 25 +/- 6.5%. Co-treatment with AS101 also partially protected against the decrease in numbers of impregnated females and litter size. AS101 increased Akt and GSK-3beta phosphorylation. CONCLUSIONS Our results indicate that AS101 can significantly protect against Cy-induced testicular damage and sperm DNA damage, probably by acting through Akt/GSK-3beta phosphorylation.

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro

Summary. An in vitro nuclear chromatin decondensation test, and quantitative nuclear ultramorphology analysis, were performed on 18 males judged to be infertile, by two failures in in vitro fertilization, and 16 fertile males. These two clinical groups only differed significantly in (1) the direction of their chromatin stability change, which took place 30–120 min post‐ejaculation while stored in the seminal plasma, and (2) in the incidence of the hypoelongated sperm‐head. Generally, the fertile male group exhibited positive chromatin stability change after prolonged storage, and low incidence of hypoelongated sperm heads, and vice versa in the unexplained infertile group. When the nuclear chromatin decondensation test and quantitative ultramorphology analysis were performed in step‐wise fashion, it was possible to correctly classify 94% of the fertile cases with 6% of false‐negative, and 89% of the unexplained infertile cases with 11% of false‐positive. Therefore, it appears that these tests might be of benefit clinically for identifying functional properties of sperm‐cells in unexplained infertile males, which cannot be detected by routine semen analysis.

Interchromosomal effect leading to an increase in aneuploidy in sperm nuclei in a man heterozygous for pericentric inversion (inv 9) and C-heterochromatin

AbstractWe describe a man with pericentric inversion 9 and constitutive heterochromatin, and a high disomy rate in his sperm cells (with all probes analyzed). The disomy rate was estimated with the following probes: 8, 9, 18, X, and Y, and was significantly higher than that in control sperm cells, while chromosome 9 showed the highest disomy frequency. The probes of X and Y together showed the same disomy frequency as X and Y alone, which indicates the same nondisjunction rate in the first meiotic division. We suggest that the interchromosomal effect found in this man differed from other findings in sperm cells of men carrying an inversion in terms of the difference in the length of the heterochromatin between the two chromosomes 9. Also, it is well known that the effect of inversion 9 with increased heterochromatin is highly variable and may even vary in members of the same family.

The influence of in vitro caffeine treatment on human sperm morphology and fertilizing capacity

After publication in the literature that in vitro caffeine treatment causes damage of the normal shape of the sperm head and thereby decreases fertilizing capacity, we carried out a clinical and electron microscopic study to determine the influence of caffeine on the fertilizing capacity and sperm cell morphology. Sixty women (with infertile husbands) underwent artificial insemination by donor with frozen/thawed semen over a period of 12 months, using randomized addition of caffeine in alternate months. Fourteen women became pregnant during the 6 months they received caffeine-treated semen, whereas only 7 pregnancies occurred during the 6 months the women received semen without caffeine. Scanning electron microscopic examinations of fresh proven donor semen showed no morphologic changes caused by the in vitro caffeine treatment. However, quantitative morphologic analysis of the frozen/thawed semen was unsatisfactory because of the freezing technique and the masking effect of the protective medium. It is concluded that in vitro caffeine treatment of fertile donor semen does not damage the spermatozoa; furthermore, it seems to improve the fertilizing capacity.