Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection.

OBJECTIVE: To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). DESIGN: Prospective, randomized study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patient's oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. INTERVENTION(S): Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. CONCLUSION(S): Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.

Sperm head's birefringence: a new criterion for sperm selection.

OBJECTIVE: To investigate the characteristics of birefringence in human sperm heads and apply polarization microscopy for sperm selection at intracytoplasmic sperm injection (ICSI). DESIGN: Prospective randomized study. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): A total of 112 male patients had birefringent sperm selected for ICSI (study group). The clinical outcome was compared with that obtained in 119 couples who underwent a conventional ICSI cycle (control group). INTERVENTION(S): The proportion of birefringent spermatozoa was evaluated before and after treatment in relation to the sperm sample quality. Embryo development and clinical outcome in the study group were compared with those in the controls. MAIN OUTCOME MEASURE(S): Proportion of birefringent sperm heads, rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): The proportion of birefringent spermatozoa was significantly higher in normospermic samples when compared with oligoasthenoteratospermic samples with no progressive motility and testicular sperm extraction samples. Although fertilization and cleavage rates did not differ between the study and control groups, in the most severe male factor condition (oligoasthenoteratospermic with no progressive motility and testicular sperm extraction), the rates of clinical pregnancy, ongoing pregnancy, and implantation were significantly higher in the study group versus the controls. CONCLUSION(S): The analysis of birefringence in the sperm head could represent both a diagnostic tool and a novel method for sperm selection.

Localization of AKAP4 and tubulin proteins in sperm with reduced motility.

AIM: To perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure. METHODS: An immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes. RESULTS: Immunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM. CONCLUSION: While the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

Cryptorchidism and semen quality: a TEM and molecular study.

Cryptorchidism is a pathological condition defined as the failure of the testis to descend into the scrotum, the location of the cryptorchid testis can be in the inguinal canal or in the prescrotal and abdominal area, sometimes resulting in atrophic seminiferous tubules. The aim of this study was to analyze semen quality of men who underwent orchidopexy for unilateral or bilateral cryptorchidism during childhood. Semen quality was investigated by light microscopy to evaluate sperm concentration and motility. Sperm morphology was performed by transmission electron microscope (TEM), and the data were mathematically elaborated. The presence of Y microdeletions was investigated by polymerase chain reaction. The effect of cryptorchidism on meiosis was explored by fluorescence in situ hybridization (FISH). The incidence of azoospermia was higher in the group with bilateral compared with unilateral cryptorchidism, and semen parameters were better in the unilateral group. Sperm pathologies detected by TEM indicated a severe deterioration of sperm quality in both groups. Necrosis and apoptosis appeared to be the most frequent pathologies, and their values reached statistical significance compared with those from fertile controls. The presence of chromosome Y microdeletions in patients with cryptorchidism and severe spermatogenetic defects is controversial. No microdeletions were found in this study. FISH values indicated that the mean percentage of gonosome disomies and diploidies were generally out of normal range, indicating a severe disturbance of meiotic segregation. The effects induced by cryptorchidism resolved in childhood seem to include a spermatogenetic impairment, leading to recommendation of detailed ultrastructural and chromosomal sperm analyses before undertaking assisted reproductive techniques.

Studies on varicocele III: ultrastructural sperm evaluation and 18, X and Y aneuploidies.

The idea that varicocele plays a detrimental role in fertility is supported by the presence of a higher frequency of affected men among the infertile population than among men with normal semen parameters. In this research we examined ejaculates from a large group of selected men affected by varicocele by light and electron microscopy. The effect of varicocele on chromosome meiotic segregation was investigated by fluorescence in situ hybridization (FISH). The potential benefits of varicocelectomy on sperm quality were evaluated by analyzing sperm characteristics before and after surgical correction of varicocele. Transmission electron microscopy (TEM) analysis, elaborated previously, showed that the incidence of immaturity, apoptosis, and necrosis was higher in the varicocele group than in controls. FISH analysis performed on sperm nuclei from selected patients with varicocele showed that the mean frequencies of disomies and diploidies were generally out of the normal range, indicating a severe disturbance in meiotic segregation. Sperm characteristics evaluated before and after varicocele repair showed a general improvement. As a consequence, the varicocele seem to affect sperm morphology and function concomitantly with meiotic segregation derangement. In consideration of these data, we suggest that TEM and FISH analyses should be performed for all varicocele patients.

Fluorescence in situ hybridization and molecular studies in infertile men with dysplasia of the fibrous sheath.

OBJECTIVE: To perform fluorescence in situ hybridization (FISH) and molecular analysis in patients with the genetic sperm defect "dysplasia of the fibrous sheath" (DFS). DESIGN: Retrospective study. SETTING: Regional Referral Center for Male Infertility, Siena, Italy. PATIENT(S): Twelve infertile patients with DFS sperm defects. INTERVENTION(S): Family history, lymphocytic karyotype, physical and hormonal assays, semen analysis. MAIN OUTCOME MEASURE(S): The DFS sperm phenotype was defined by light, fluorescent, and electron microscopy. Sperm chromosomal constitution was examined by FISH. Gene deletions were tested by polymerase chain reaction. RESULT(S): The genetic sperm defect DFS was determined by transmission and scanning electron microscopy. Immunofluorescence staining of A-kinase anchoring protein 4 (AKAP4) showed a moderate and diffuse signal, revealing a disorganized and incompletely assembled fibrous sheath. In 11 of 12 DFS patients, polymerase chain reaction for detecting the presence of partial sequence of AKAP4/AKAP3 binding regions gave positive results. Fluorescence in situ hybridization was performed in decondensed sperm nuclei with probes for chromosomes 18, X, and Y. The mean disomy frequency of chromosome 18 was in the normal range, whereas the mean disomy frequencies of sex chromosomes and diploidies were twice those of controls. CONCLUSION(S): These results should be considered when DFS sperm are used in assisted reproductive technology, owing to the high risk of transmission of chromosomal unbalance and of DFS sperm defects to male offspring.

Quantitative evaluation of spermatozoa ultrastructure after acupuncture treatment for idiopathic male infertility.

OBJECTIVE: To evaluate the ultramorphologic sperm features of idiopathic infertile men after acupuncture therapy. DESIGN: Prospective controlled study. SETTING: Christian-Lauritzen-Institut, Ulm, IVF center Munich, Germany, and Department of General Biology, University of Siena, Siena, Italy. PATIENT(S): Forty men with idiopathic oligospermia, asthenospermia, or teratozoospermia. INTERVENTION(S): Twenty eight of the patients received acupuncture twice a week over a period of 5 weeks. The samples from the treatment group were randomized with semen samples from the 12 men in the untreated control group. MAIN OUTCOME MEASURE(S): Quantitative analysis by transmission electron microscopy (TEM) was used to evaluate the samples, using the mathematical formula based on submicroscopic characteristics. RESULT(S): Statistical evaluation of the TEM data showed a statistically significant increase after acupuncture in the percentage and number of sperm without ultrastructural defects in the total ejaculates. A statistically significant improvement was detected in acrosome position and shape, nuclear shape, axonemal pattern and shape, and accessory fibers of sperm organelles. However, specific sperm pathologies in the form of apoptosis, immaturity, and necrosis showed no statistically significant changes between the control and treatment groups before and after treatment. CONCLUSION(S): The treatment of idiopathic male infertility could benefit from employing acupuncture. A general improvement of sperm quality, specifically in the ultrastructural integrity of spermatozoa, was seen after acupuncture, although we did not identify specific sperm pathologies that could be particularly sensitive to this therapy.

An ultrastructural and immunocytochemical study of a rare genetic sperm tail defect that causes infertility in humans.

OBJECTIVE: To characterize and describe the ontogenesis of a rare flagellar defect affecting the whole sperm population of a sterile man. DESIGN: Case report. SETTING: Regional referral center for male infertility in Siena, Italy. PATIENT(S): A 28-year-old man with severe asthenozoospermia. INTERVENTION(S): Physical and hormonal assays, semen analysis, and testicular biopsy. MAIN OUTCOME MEASURE(S): Semen samples and testicular biopsies were analyzed by light and transmission electron microscopy; immunocytochemical study with anti-beta-tubulin and anti-AKAP 82 antibodies was performed to detect the presence and distribution of proteins. RESULT(S): Ultrastructural analysis of ejaculated spermatozoa and testicular biopsy revealed absence of the fibrous sheath in the principal-piece region of the tail. Fibrous sheath-like structures were observed in cytoplasmic residues and residual bodies released by spermatids in the seminiferous epithelium. Other anomalies observed were supplementary axonemes and mitochondrial helix elongation. These features were confirmed by immunocytochemical staining. CONCLUSION(S): This rare sperm tail defect, characterized by absence of the fibrous sheath, presence of supplementary axonemes, and an abnormally elongated midpiece, originates in the seminiferous tubules during spermiogenesis, as detected in testicular biopsy sections. These defects occur in the whole sperm population, and therefore a genetic origin could be suggested.

Infertile spermatozoa in a human carrier of robertsonian translocation 14;22.

OBJECTIVE: To present the ultrastructural, functional, and chromosomal analyses of spermatozoa from an infertile man with normal phenotype and chromosomal translocation 14;22. DESIGN: Case report. SETTING: Regional Reference Center for Male Infertility in Siena, Italy. PATIENT(S): A 36-year-old man with primary infertility for 3 years and his parents. INTERVENTION(S): Family history and lymphocytic karyotypes, physical and hormonal assays, and semen analysis. MAIN OUTCOME MEASURE(S): Morphological sperm evaluation was performed by light, fluorescent, and electron microscopy; chromosomal constitution was examined by the fluorescence in situ hybridization (FISH) technique. The penetration ability of spermatozoa was checked by the hamster test. RESULT(S): The spermatozoa of the patient showed unusual ultrastructural defects. The nuclei were large, spheroidal, and generally uncondensed; the acrosomes were frequently absent or reduced; and the axonemes were often devoid of dynein arms or central singlet tubules. These characteristics are related to immaturity. The lymphocytic karyotype revealed a robertsonian translocation 14;22 in the sterile patient and his mother. FISH sperm analysis demonstrated a high frequency of diploidy for the chromosome 18,XY. The hamster penetration test gave negative results. CONCLUSION(S): The unusual structural sperm immaturity is associated with the translocation 14;22. This chromosomal anomaly may therefore negatively influence the spermatogenesis; an interchromosomal effect on meiosis segregation is also suggested.

Insulin-dependent diabetes in men is associated with hypothalamo-pituitary derangement and with impairment in semen quality.

BACKGROUND: The objective of the study was to investigate the hypothalamo-pituitary-testicular axis and sperm structure at the transmission electron microscope (TEM) level in men affected by insulin-dependent diabetes. METHODS: Twenty-two diabetic men and 24 controls were recruited. GnRH (100 micro g) was administered and FSH- and LH-induced secretion was evaluated. Semen samples were collected and sperm concentration and motility were determined using a Makler chamber. Ejaculated sperm were fixed and observed with a TEM. RESULTS: The response of gonadotrophins to GnRH was significantly lower in diabetics than in control men. Sperm motility was also significantly lower. At the electron microscope level, sperm from diabetics exhibited a higher percentage of immaturity- and apoptosis-related defects than sperm from controls. CONCLUSIONS: The reduced response of gonadotrophins to GnRH in diabetic men may indicate a decreased acute releasable pool of pituitary gonadotrophins. The results of TEM examination showed that sperm from men with diabetes presented severe structural defects in comparison with sperm from controls. It is possible that the reproductive impairment recognized in men with diabetes could be the result of interference by the disease on the hypothalamo-pituitary-testicular axis at multiple levels, as indicated by the reduced gonadotrophin response to appropriate stimuli and by the abnormal ultrastructure of ejaculated sperm. The defective spermatogenesis may be the consequence of a direct testicular effect of the disease.