Morphological alterations in protamine-deficient spermatozoa.

STUDY QUESTION: How are protamine deficiencies associated with sperm head morphology in subfertile men? SUMMARY ANSWER: The prevalence of morphological variations and large nuclear vacuoles was slightly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa. WHAT IS KNOWN ALREADY: A protamine deficiency was previously reported to be associated with an abnormal sperm morphology; however, how they are related to each other remains unclear. This is further confounded by a number of protamine-deficient spermatozoa having a normal head morphology. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study, including 36 men diagnosed with male factor infertility or participating in an assisted reproduction program. To assess sperm head morphology, this study analyzed 2400 spermatozoa with a protamine deficiency and 2400 spermatozoa with a normal protamine status. An additional 21 men were analyzed to examine DNA fragmentation and its relationship with protamine deficiencies and sperm head morphologies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The morphology of the sperm head was evaluated based on its shape, size and nuclear vacuoles at a magnification of >6000×. Using elliptic Fourier analysis, the shape was summarized into four numeric variables. The protamine status was evaluated with chromomycin A3 (CMA3). Sperm head size, vacuoles and shape were compared between protamine-deficient and non-deficient spermatozoa. DNA fragmentation was evaluated with the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. The percentages of protamine-deficient spermatozoa and DNA fragmentation were compared between spermatozoa with morphologically normal heads and those with abnormal heads. MAIN RESULTS AND THE ROLE OF CHANCE: Variations in head size (P < 0.0001) and shape (P < 0.0001) were significantly higher, with narrower (P < 0.001), more fan-shaped (P < 0.01) and more square-shaped forms (P < 0.001) in protamine-deficient spermatozoa than in non-deficient spermatozoa; however, the distribution of morphological variations markedly overlapped. Protamine deficiencies were more frequently observed in spermatozoa with large nuclear vacuoles than in those without them (32.0 ± 3.1 versus 39.4 ± 2.9%, P < 0.001). The percentage of protamine-deficient spermatozoa was significantly lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (25.4 ± 2.6 versus 38.0 ± 2.5%, P < 0.001). The percentage of DNA fragmentation was significantly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa (11.3 ± 2.1 versus 1.6 ± 0.6%, P < 0.001), and was lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (2.6 ± 0.7 versus 6.4 ± 0.2%, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: We were unable to discriminate the kind of protamines or quantify the extent of the protamine deficiency in spermatozoa using the CMA3 staining method. WIDER IMPLICATIONS OF THE FINDINGS: This study provided a novel insight into how abnormal protamination affects sperm head morphology as well as the relationship between sperm head morphology and its own molecular integrity. Our results will contribute to a deeper understanding of the benefits and limitations of the morphological selection of spermatozoa for ICSI. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a JSPS Grant-in-Aid for the Encouragement of Scientists (25931009, 26931010). All authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Enantiomorphs differ in shape in opposite directions between populations.

Development is left-right reversed between dextral and sinistral morphs of snails. In sympatry, they share the same gene pool, including polygenes for shell shape. Nevertheless, their shell shapes are not the mirror images of each other. This triggered a debate between hypotheses that argue either for a developmental constraint or for zygotic pleiotropic effects of the polarity gene. We found that dextrals can be wider or narrower than sinistrals depending on the population, contrary to the prediction of invariable deviation under a developmental constraint. If the pleiotropy is solely responsible instead, the mean shape of each morph should change, depending on the frequency of polarity genotype. Our simulations of this mean shape change under zygotic pleiotropy, however, show that the direction of interchiral difference remains the same regardless of genotype frequency. Our results suggest the presence of genetic variation among populations that changes the maternal or zygotic pleiotropic effect of the polarity gene.

Quantitative analysis of stromal fat content of human parathyroid glands associated with thyroid diseases using computer image analysis.

The stromal fat content in the parathyroid glands and its significance are still controversial. Several methods have also been introduced to evaluate the content in the literature. In an attempt to better understand the significance of stromal fat content, a computer image analyzing system was applied and 62 glands were investigated using routinely processed tissue. Our study revealed that the average stromal fat content was 34.8 +/- 18.7% which was lower than that of other studies. The stromal fat content was correlated with the age, grade of obesity, area of the gland and serum parathormone level; however, no consistent patterns were obtained with other clinical parameters. These results indicate that the change of stromal fat content may be related to the general condition of the patient similar to the change of fat in other organs. However, the decrease of the content may indicate the early change of parathormone secretory activity even if the serum examination shows no evidence of hyperfunction of the glands.

[HPV infection of the uterine cervix by cytodiagnosis, histodiagnosis and dot blot hybridization method].

Human papilloma virus (HPV) infection is diagnosed by morphological methods (including cytodiagnosis, histodiagnosis and electron microscopy) and DNA diagnostic methods. One of the DNA diagnostic methods, Dot blot hybridization (DBH), is generally regarded to be superior to the morphological methods in terms of specificity and sensitivity. There have recently been reports showing that such morphological methods as cytodiagnosis [for koilocytotic atypia (KC) or koilocytosis] and histodiagnosis are best for HPV detection in laboratories because they have better sensitivity, and are cheaper, and procedurally simpler than the DBH method. We compared detection rates by morphological methods (cytodiagnosis and histodiagnosis) and the DBH method. (Materials and Methods) The subjects were 377 patients who came to the Department of Obstetrics and Gynecology of Juntendo University Urayasu Hospital and had abnormal findings in the uterine cervix by cytodiagnosis of colposcopy. Uterine cervix swabs were scraped during colposcopic examination, and specimens for Papanicolaou and Giemsa staining were prepared on 2 slides. Scraping was again performed for the DBH (Vira pap. and type, Toray Industries, Inc.) method using swabs specified for the kit, and cells were obtained in a collection kit. At the same time, at least 3 sites including, possible atypical conversion zones, were biopsied, followed by paraffin embedding and hematoxylin-eosin staining of the specimens. (Results) 1) Comparison of detection rates by cytodiagnosis and the DBH method The positive rates by the DBH method were determined according to cytodiagnostic category in 377 patients. HPV-DNA was detected in every category and in 86 (22.8%) of all of the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of HPV infection in CIN and invasive cancers by in situ hybridization method--comparison with histopathology].

The causal association of human papilloma virus (HPV) with cervical cancer has been supported by multiple lines of evidence. Therefore, in the case of dysplasia, the presence of HPV-DNA should be detected and its subtypes identified. This is important in the determination of the prognosis for cervical disease. We reported a study in which the localization and types of HPV in cervical diseases was identified by in situ hybridization using biotin-labelled DNA probes. Seven types (3 basic forms) of HPV were used as DNA probes. HPV types used were following: 6/11, 16/18, 31/33/35. In this study, we introduced 7 new types (3 forms) of probes for a total of 14 types (6 forms). The new probes introduced were 42/43/45, 45/56, 51/52. Using these probes, the rate of detection HPV according to types was examined. In addition, localization of HPV infection and its relationship with histopathological findings of cervical disease were evaluated. 1) Types 6/11 and 45/56 were found in the lesions with less important histological findings (CIN I). Types 16/18, 31/33/35, 51/52 were found in the all lesions. Type 42/43/44 were not detected in this study. 2) The addition of 7 new types (3 new forms) of probes resulted in a 17% increase (16 cases) in the rate of detection of HPV compared to our previous study. 3) Localization and distribution pattern of the HPV is not, as has been reported so far, dependent on HPV type but rather on the histological characteristics, such as the degree of dysplasia.

[81mKr scintigraphic evaluation of hemodynamics in gynecologic malignancies under condition of angiotensin II-induced hypertension].

Transcatheter arterial infusion chemotherapy is one of the most useful therapeutic procedures for gynecologic malignancies. Recently, several reports have been published about Angiotensin II-induced hypertension chemotherapy and the efficacy of the method, but there have been no reports to evaluate an application for gynecologic malignancies. We evaluate the usefulness of the method for gynecologic malignancies demonstrating the changes of hemodynamics of the tumor using 81mKr scintigraphy. Thirteen patients with pathologically confirmed gynecologic malignancies were evaluated by angiography and continuous infusion of 81mKr via the catheter with and without Angiotensin II. At first, continuous infusion of 81mKr was performed under the superselective catheterization of the uterine artery. The radioactivities in the ROI were counted. Then, withdrew the catheter from the uterine artery to the internal iliac artery, and again continuously infused 81mKr and counted the radioactivities in the same ROI. Finally, keeping the catheter in the internal iliac artery, Angiotensin II and 81mKr were infused simultaneously. And counted the radioactivities. The radioactivities were highest when the catheter tip was placed in uterine arteries and lowest when the catheter tip was placed in internal iliac arteries. But radioactivities in the ROIs were definitely increased when Angiotensin II was used, even if the catheter tip was keeping in the internal iliac arteries. The optimal catheter position of transcatheter arterial chemotherapy for gynecologic malignancies is at proximal uterine artery. Since Angiotensin II-induced hypertension may increase blood flow of tumors, it seems to have indication for post-operative cases, highly advanced cases and cases with difficulties to perform superselective catheterization.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of papilloma virus of the human uterine cervix by in situ hybridization method--comparison with immunohistochemistry].

The usual methods for pathological diagnosis of HPV infection of the uterine cervix include screening in cytodiagnosis and histodiagnosis and confirmation by immunohistochemistry (IHC) method. However, some institutes have recently begun to use in situ hybridization (ISH) method for definitive diagnosis using a DNA probe. We compared IHC with ISH with regards to the localization and rate of detection of HPV in lesions of the uterine cervix such as dysplasia and squamous cell carcinoma in the present study. The cases found positive by IHC showed brownish nuclei of the epithelium and those positive in ISH showed purple to purplish-black nuclei. The comparison of cases positive by both methods revealed that the number of cells positive by IHC was smaller than that by ISH, and the cells positive by IHC were localized in the superficial layer. HPV was detected by the IHC various lesions of the uterine cervix in 13 (12.3%) of 106 patients, while it was detected by the ISH in 39 (36.8%) of 106 patients. The results of both methods were in accordance in 66.0% (77 patients; positively in 8 and negatively in 62). The detection sensitivity of IHC is lower than that of ISH. IHC cannot be used to identify the type of HPV, and it is impossible to confirm the presence or absence of virus by this method in cases of malignant changes. ISH is therefore necessary for identification of HPV and investigation of a histopathological relationship between HPV type and malignant change.

[Diagnosis and follow-up study of human papillomavirus infection in benign epithelium and mild dysplasia of the uterine cervix].

In this study, we evaluated the sensitivity of cytology, colposcopy and the dot blot method (Vira Pap, Vira Type) for the screening of HPV infection in benign epithelium and mild dysplasia of the uterine cervix, and then investigated subsequent changes in HPV DNA in the lesion. The HPV infection detection rates for cytology, colposcopy and the dot blot method were 14/46 (30.4%), 30/46 (65.2%) and 35/46 (76.1%), respectively. Cases where HPV DNA was negative with the dot blot method but HPV infection was suspected with cytologic and colposcopic examination were 3/6 (50.0%) in the undigestive type except for HPV types 6, 11, 16, 18, 31, 33 and 35. HPV DNA disappeared after 3-6 months in persistent cases with histologically benign epithelium in the one year follow-up. In persistent cases of mild dysplasia followed up histologically for one year from the time of the first visit showed differences between negative and positive cases of HPV DNA after 1 year. In 2 progressive cases (HPV 31 and 16 types) HPV DNA in the cervix was detected continuously. We consider that the dot blot technique is a valuable method for identifying human papillomavirus infection. However, detection of undigestive HPV types by the dot blot method was poor. Therefore, the joint use of cytology, colposcopy and the dot blot method seems to be a valuable screening technique. Individual variations in the clinical course of the high risk group appear to reflect profound differences in the effectiveness of host responses. Therefore, cases where continuous HPV DNA was observed in the high risk groups should be followed up for a long period.

[Detection of HPV-DNA in the various uterocervical lesions by in situ hybridization].

We detected HPV (Human Papilloma Virus) -DNA in various uterocervical lesions by in situ hybridization using biotinylated DNA probes. In cases positive for HPV, the nuclei of the epithelial cells was purple to blackish purple. In 2 of 6 cases of chronic cervicitis, HPV-DNA was detected in the outer layer of the squamous epithelium. Eleven of 19 with mild dysplasia (57.9%) showed a positive reaction in the upper one-third of the epidermis in a mainly consisting of koilocytotic cells. All 6 patients with moderate dysplasia had positive cells among the koilocytotic cells and atypical cells in the middle layer. Five of 11 patients with severe dysplasia had scattered positive cells. Two of them had atypical condylomatoid lesions. Eight of 32 patients with squamous cell carcinoma were positive for HPV-DNA, but there was no consistent distribution pattern of the positive cells.

[Detection of papilloma virus in the cervix of the human uterus by in situ hybridization--Comparative study of detection rate by Southern blot method].

Cases found to be positive for human papilloma virus (HPV) infection in the cervix by Southern blot method were evaluated with a newly developed kit for in situ HPV tissue hybridization. By the in situ hybridization method, HPV-DNA was detected without damaging the tissue structure. It appeared as purple or black stains in the nucleus of the epithelial cells that were located at the level of one-third of the epithelial thickness from the surface. The cases that were negative with the Southern blot method were also negative with the in situ hybridization method, but only 55.8% of the cases positive with the Southern blot method were positive with the in situ hybridization. Although in situ hybridization method is not as sensitive as the Southern blot method, it allows analysis of old paraffin blocks and comparison with pathological features.

[Fundamental and clinical studies on human papillomavirus infection in the uterine cervix especially by Vira Pap (dot blot) method].

Female genital organs infected with human papillomavirus (HPV) have drawn attention as STD and in connection with the mechanism of carcinogenesis. Recently, we used a simplified HPV detection kit, Vira Pap method in clinical tests. To estimate the usefulness of the Vira Pap method in clinical application, a comparison was made between the results of the Vira Pap method and those of the conventional method. Furthermore, an investigation was made of the relationship between uterine-cervical lesions and the types of HPV. The following findings were obtained: 1. It was demonstrated that the Vira Pap method was superior to the conventional methods and was almost equal to the Southern blot method. 2. The cases in which HPV infection were confirmed by the Vira Pap method were further analyzed and HPV typed by the Southern blot method. And, types 6, 11, 18, 31, 33, 35 and unclassifiable types were found. 3. HPV 16, 18, and 33, which are said to be closely related to carcinoma, were detected in cases of chronic cervicitis as well. In these cases, further investigation seems to be required.