Immunologically reactive albumin-like protein in human testis and seminal plasma

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

Predictive value of DNA cytometry in CIN 1 and 2. Image analysis of 193 cases

OBJECTIVE: To investigate DNA image cytometry for predicting the prognosis of cervical intraepithelial neoplasia (CIN). STUDY DESIGN: Smears from 151 women affected by CIN 1 or 2 on cytology with minimal follow-up of three years were included. Sixty-seven showed progression, with histologically confirmed carcinoma in situ or invasive cancer. Eighty-four cases showed regression of the disease, which was cytologically, histologically and colposcopically confirmed. Papanicolaou-stained smears were destained, and the Feulgen reaction was performed with consecutive image DNA cytometry of suspicious cells using an image analysis system (Cires, Zeiss, Germany). The DNA index of the greatest stemline and the number of single aneuploid cells, using 9c exceeding events, were computed. RESULTS: In the group with progression, an aneuploid DNA stemline was found in 25 smears (26.9%). In 64 cases (66.7%) more than one aneuploid event was detected. The total number of aneuploid cases in this group was 76 (81%). In the group without progression, the number of aneuploid stemlines was 2 (2%). Single aneuploid cells could be found in five cases (5%). The overall number of aneuploid cases in that group was five. The sensitivity was 74.3%, positive predictive value 85.2% and negative predictive value 77%. CONCLUSION: Aneuploidy is a marker for prospective malignancy in cervical Papanicolaou smears. DNA image cytometry, as an additional method, can be used to predict outcome in patients with CIN 1 and 2 of the cervix. DNA cytometry is not a screening method but can add further information for a treatment decision in doubtful cases.     

Prevalence of carcinoma in situ of the testis in 207 oligozoospermic men from infertile couples: prospective study of testicular biopsies.

OBJECTIVE: To investigate the prevalence of carcinoma in situ of the testis in a group of oligozoospermic men from infertile couples. DESIGN: A consecutive group of oligozoospermic men from infertile couples were offered bilateral testicular biopsy. The observed prevalence of carcinoma in situ was compared with the expected prevalence of testicular cancer in a corresponding age matched population of Danish men, assuming all untreated cases of carcinoma in situ progress to tumour stage. This calculation was based on data from the Danish Cancer Registry. SUBJECTS: 207 men aged 18-50 years who had sperm density below 10 million/ml in two samples within the previous 2 years or sperm density below 20 million/ml in two samples within the previous 2 years and a history of cryptorchidism or one or two atrophic testicles (orchidometer volume less than 15 cm3), or both. INTERVENTIONS: Bilateral testicular biopsies. MAIN OUTCOME MEASURES: Carcinoma in situ in the biopsy specimen. RESULTS: No case of carcinoma in situ was found among the 207 men. The expected number in a normal age matched population of corresponding size was 0.8. CONCLUSIONS: There is no increase in risk of carcinoma in situ of the testis in moderately oligozoospermic men of couples referred because of infertility.     

DNA profiles in dysplasia and carcinoma of the human esophagus

The nuclear DNA content was microspectrophotometrically measured in 16 resected esophagi having dysplasia, carcinoma in situ and/or early invasive squamous carcinoma. First, the epithelial thickness in (1) normal squamous esophageal epithelium and (2) dysplasia-carcinoma in situ areas was divided into three equal compartments (i.e., basal-parabasal, intermediate and superficial) in five cases. In the normal epithelium, while some of the nuclei in basal-parabasal normal squamous cells had elevated DNA values (corresponding to the natural DNA replication in these cells), intermediate and superficial (nonreplicating) normal squamous cells showed a more definite clustering about the 2c value. In the nonnormal epithelium, the percentage of cells with DNA levels exceeding the normal tetraploid value was highest for the intermediate zone. Therefore, in all 16 cases, normal intermediate cells were measured as internal controls, against which the DNA levels of cells in the intermediate compartment in the areas of dysplasia and/or carcinoma in situ were compared. In areas of dysplasia, two different DNA patterns were observed: one clustering around the normal diploid region and the other with aneuploid values. While the former corresponded to some of the lesions considered by conventional histologic examination to be slight and moderate dysplasias, the aneuploid pattern corresponded to the remaining slight and moderate dysplasias as well as to the severe dysplasias. The possibility that "diploid dysplasias" are reactive (i.e., nonneoplastic) lesions due to chronic inflammation or are "dormant" nonprogressive dysplasias, while aneuploid dysplasias are more aggressive lesions, seems to be substantiated by the fact that all areas with carcinoma in situ or with microinvasive squamous carcinoma had aneuploid nuclei.     

Immunocytochemical localization of DJ-1 in human male reproductive tissue

DJ-1 was identified as an activated ras-dependent oncogene product, and was also found to be an infertility-related protein (contraception-associated protein 1; CAP 1) that was reduced in rat spermatozoa treated with ornidazole, one of the endocrine disrupting substances that causes reversible infertility in rats. CAP 1 is present in spermatozoa but is not detectable in the epididymal fluid of fertile rats and appears to be shed from sperm during treatment with ornidazole. To determine the functions of DJ-1 in the human reproductive system as a target protein of endocrine active substances, we identified the localization of DJ-1 in human testis, epididymis, ejaculated spermatozoa, and seminal plasma. DJ-1 was present in cells existing in the seminiferous tubules and Leydig cells. Some strong expressions were observed in Leydig cells and Sertoli cells, suggesting a relation with spermatogenesis via androgen receptor (AR). In ejaculated spermatozoa, DJ-1 existed on the surface of the posterior part of head and the anterior part of the midpiece. DJ-1 was also present on sperm flagella when the antibody penetrated the plasma membrane, suggesting that there are two putative roles in fertilization, one is binding to the egg, and the other is flagella movement. In contrast to previous findings, we detected DJ-1 in seminal plasma of fertile men. These results demonstrate that DJ-1 in human seminal plasma is not only from spermatozoa but also from the testis and epididymis. It is suggested that DJ-1 may play an important and as yet uncharacterized role in spermatogenesis and fertilization in humans.     

Ascaris suum: electrophoretic characterization of reproductive tract and perienteric fluid polypeptides, and effects of seminal and uterine fluids on spermiogenesis.

Fluids isolated from the testis, seminal vesicle, uterus, and pseudocoelomic cavity of Ascaris suum were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured for protein concentration, pH, and osmolarity. The testis and seminal fluids display much homology and share major polypeptide components having molecular weights of 15,000 and 35,000. A cytoplasmic extract of spermatids from the seminal vesicle exhibited a banding pattern nearly identical to that of testis fluid. The seminal fluid has unique major components of 57,000 and 150,000, and seminal fluid from individual worms showed differences in major band concentration and distribution of minor components. The uterine fluid has major polypeptides of 14,000, 16,000, 66,000, 74,000, 120,000, and 140,000, and exhibits more similarity to the perienteric fluid then either the seminal or testis fluids. Electrophoretic comparisons of four uterine regions revealed nearly identical banding patterns although somewhat higher concentrations of four major components occurred in certain segments. The male and female perienteric fluids have major bands at 40,000, 120,000, and 140,000, and the female fluid has more intense minor components of 90,000 and 115,000. Perienteric fluid from individual worms differed only in minor band distribution. The reproductive fluids have numerous minor components mostly from 20,000 to 70,000, while the perienteric fluid minor bands are mainly located in the 80,000 to 120,000 range. The pH of the seminal fluid (6.5) differs from that of the uterine fluid (7.7), and both seminal and uterine fluids are of lower osmolarity than the perienteric fluid. In vitro studies demonstrate that uterine fluid does not induce spermatid transformation into bipolar, ameboid spermatozoa, while the seminal fluid induces only lipid granule coalescence in either seminal vesicle or terminal testis spermatids.     

Cryptorchidism: aspects of fertility and neoplasms. A study including data of 1,335 consecutive boys who underwent testicular biopsy simultaneously with surgery for cryptorchidism.

PURPOSE: An attempt to make a rational strategy for treatment of cryptorchidism. MATERIALS AND METHODS: 1,335 cryptorchid boys with biopsy at surgery (1,638 specimens). We studied: frequency of no germ cells in biopsies from 698 patients <12 years at surgery; fertility potential of 140 patients who were now adults, and apperance of testicular neoplasia in all biopsies. RESULTS: Lack of germ cells appeared from 18 months. The frequency increased with increasing age. It appeared in 30% (61/202) bilateral, and 18% (88/496) unilateral cases. In men who had undergone bilateral or unilateral orchiopexy, respectively, there was normal sperm count in 19% (14/75) and 83% (54/65), and infertility was suspected in 56% (42/75) and 8% (5/65) (FE, p < 0.00005, p < 0.00005), respectively. The lowest, the mean, and the highest age-matched spermatogonia count per tubule at orchiopexy was associated with sperm count (Spearman test, p < 0.0001, p < 0.005, p < 0.05). Isolated, this was demonstrated for the 75 formerly bilateral (Spearman, p < 0.0001, p < 0.0001, p < 0.0001), but not the 65 formerly unilateral cases (Spearman, p = 1.0). No germ cells at orchiopexy was associated with suspected infertility. Risk was 78-100% in bilateral (dependent on one or both testes affected), and 33% in unilateral cryptorchidism. There was one invasive germ cell tumor, six cases of carcinoma in situ testis, and one Sertoli cell tumor. Three neoplasms were diagnosed in intra-abdominal testes, four in boys with abnormal external genitalia, and two in boys with known abnormal karyotype. Risk of neoplasia was 5% (7/150) in patients with intra-abdominal testis, abnormal external genitalia or diagnosed abnormal karyotype, versus 0% (0/1,185) in patients without these characteristics (FE, p < 0.00005). CONCLUSION: We recommend surgery for cryptorchidism before 15-18 months of age because: (a) lack of germ cells is very rare before, and (b) lack of germ cells is associated with subsequent risk of infertility. At primary surgery for cryptorchidism, we recommend examination for testicular neoplasia in cases of intra-abdominal testis, abnormal external genitalia or known abnormal karyotype.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

Oxytocin in the semen and gonads of the stallion

It has been suggested that oxytocin is involved in sperm transport and motility in domestic animals. Immunoreactive oxytocin was measured in seminal fractions (pre-ejaculatory fluid, seminal plasma, gel and sperm) and in extracts of testis and epididymis from stallions. In addition, sections of gonadal tissue from stallions were immunostained for the presence of oxytocin and its neurophysin. Oxytocin was detected in all of the seminal fractions, being highest in the gel. It was also present in washed, lysed sperm and in extracts from the testis and epididymis. Immunostaining for oxytocin was present in occasional interstitial cells in the testis and in the epididymal epithelium and smooth muscle. However, immunostaining for neurophysin was detected in a few interstitial cells in the testis of only 1 of 8 stallions and was absent from all areas of the epididymis. These data demonstrate for the first time the presence of oxytocin in stallion semen and gonadal tissue; however, lack of immunostaining for neurophysin indicated that it was unlikely that there was local synthesis within the gonads.     

Seminal fluid excretion of cytomegalovirus related to immunosuppression in homosexual men.

Seminal fluid samples from 84 Danish homosexual men were successfully cultured to determine the prevalence of cytomegalovirus excretion. Ten (15%) out of 66 men positive for the antibody were found to be excreting the virus. Although the proportion excreting was inversely related to age (p less than 0.01), three men aged over 30 and with many years of homosexual experience excreted the virus. In addition, a 50 year old man with Kaposi''s sarcoma excreted the virus. A further study of the ratio of T cell helpers to suppressors in the men aged over 30 and a series of age matched non-excreting homosexual control or heterosexual men showed that those excreting cytomegalovirus in their seminal fluid had statistically lower ratios (all less than 0.77) than the controls (p less than 0.05). Excretion of cytomegalovirus may be related to re-emergence of latent infection in immunosuppressed homosexual men.     

Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system

Seminal plasma is a fluid that originates from the testis, epididymis,prostate, and seminal vesicles, and hence, proteomic studies may identify potential markers of infertility and other diseases of the genito-urinary tract. We profiled the proteomes of pooled seminal plasma from fertile Control and post-vasectomy (PV) men. PV seminal plasma samples are void of proteins originating from the testis and the epididymis due to ligation of the vas deferens, and hence, comparative analysis of Control and PV data sets allows for identification of proteins originating from these tissues. Utilizing offline MudPIT and high-resolution mass spectrometry, we were able to identify over 2000 proteins in Control and PV pools each and over 2300 proteins all together. With semiquantitative analysis using spectral counting, we catalogued 32 proteins unique to Control, 49 at lower abundance in PV, 3 unique to PV, and 25 at higher abundance in PV. We believe that proteins unique to Control or at lower abundance in PV have their origin in the testis and the epididymis. Public databases have confirmed that many of these proteins originate from the testis and epididymis and are linked to the reproductive tract. These proteins may serve as candidate biomarkers for future studies of infertility and urogenital diseases.     

Screening for carcinoma in situ of the contralateral testis in patients with germinal testicular cancer.

Two hundred and fifty biopsy specimens from the contralateral testis in patients with unilateral germinal testicular cancer were analysed by light microscopy for carcinoma-in-situ changes. Changes were found in 13 (5.2%) patients. One-third of patients with an atrophic contralateral testis (volume less than or equal to 12 ml) and one-fifth of patients with a history of cryptorchidism had changes in the remaining testis. In the present series 85% of cases with carcinoma-in-situ changes would have been diagnosed if the one-fifth of the patients having an atrophic testis or a history of cryptorchidism or both had been screened. Since the natural course of carcinoma in situ in the contralateral testis of patients with germinal testicular cancer has not been established, the patients are being re-evaluated frequently. To date two patients with carcinoma in situ have developed a second cancer.     

Clone heterogeneity in diploid and aneuploid breast carcinomas as detected by FISH

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.     

Nuclear DNA analysis of koilocytic and premalignant lesions of the uterine cervix.

Cervical biopsy samples were taken from 79 patients who had various grades of cervical intraepithelial neoplasia or who showed evidence, in the form of koilocytosis, of human papillomavirus infection of the uterine cervix and from 10 women with normal cervices. The DNA content of the cells in the samples was analysed by flow cytometry. Analysis of the data obtained showed that the biopsy samples from women with cervical intraepithelial neoplasia and human papillomavirus lesions contained significantly more dividing cells (31.2% of cells from human papillomavirus lesions with no cervical intraepithelial neoplasia and 33.06%, 29.89%, and 31.76% of cells from cervical intraepithelial neoplasia grades I, II, and III, respectively) than those from women with normal cervices (21.6%). The proportion of aneuploid samples from the group who showed evidence of human papillomavirus infection only (18.2%) did not differ significantly from the group with cervical intraepithelial neoplasia grade III (21.2%). Aneuploidy and an increased rate of cellular proliferation are recognised characteristics of malignancy. These results therefore support the view that human papillomavirus plays an important part in the aetiology of cervical carcinoma and are relevant to the clinical management of patients.     

Seminal antioxidants in humans: preoperative and postoperative evaluation of coenzyme Q10 in varicocele patients.

Coenzyme Q10 in seminal fluid shows a direct correlation with seminal parameters except in patients with varicocele. To evaluate whether surgical treatment of varicocele could revert CoQ10 abnormalities, we have studied CoQ10 distribution in thirty-three VAR patients, before and 6-8 months after varicocelectomy, twenty patients with idiopathic oligozoospermia, eleven with isolated asthenozoospermia and sixteen normal fertile men. CoQ10 was assayed in total seminal fluid, plasma or cell pellet by HPLC. A significantly higher CoQ10 proportion in seminal plasma in VAR vs. controls (mean +/- SEM: 61.68 +/- 2.41 vs. 41.60 +/- 1.99%, respectively) was present; total CoQ10 correlated with sperm motility in controls, but not in VAR; an inverse correlation between cellular CoQ10 and motility was present in VAR, but not in controls. Postoperatively, a partial reversion was observed, since the plasma-to-total CoQ10 ratio decreased, but the correlation between total CoQ10 and motility was not restored. On the contrary, the peculiar correlation between cellular CoQ10 and motility was no more detectable in postoperative VAR patients. A partial postoperative reversal of abnormalities in CoQ10 distribution and correlation with seminal parameters was therefore present. As seminal plasma CoQ10 reflects an interchange between intracellular and extracellular compartments, its different distribution could cause a greater sensitivity to peroxidative damage and a reduced utilization for energetic purpose.     

Anaerobic seminal fluid micro-flora in chronic prostatitis/chronic pelvic pain syndrome patients

We investigated the seminal micro-flora of 116 men. Eighty-four men had chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and 34 of them were also leukocytospermic. Thirty-two asymptomatic men formed the control group. Micro-organisms were found in all of the 116 seminal fluid specimens. More than 20 different micro-organisms were found in both groups. Neisseria gonorrhoeae and Chlamydia trachomatis were not found. A high frequency of anaerobic bacteria was found in all groups (68-79%), and in most of the specimens, anaerobic micro-organisms were equal to or outnumbered the aerobic strains. We found 1-8 different micro-organisms in each semen sample, the total count of micro-organisms ranged from 10(2) to 10(7)/mL of semen. Both parameters were significantly higher in leukocytospermic CP/CPPS (NIH IIIA category) patients (median=5 different micro-organisms; total median count 5 x 10(4)) than in the control group (median=3 different micro-organisms; total median count 10(3)). In the CP/CPPS patients, the prevalence and/or count of some opportunistic bacteria was higher than in the control group. To show that the micro-organisms do not originate from the urethra, first voided urine was also investigated in 17 prostatitis patients and 15 controls. One patient had significantly fewer micro-organisms (median 1 vs. 4) and a lower total count of micro-organisms (median 10(2) vs. 10(4)/mL) in the first-catch urine than in the seminal fluid. We found only one third of the micro-organisms to be similar in urine and semen while anaerobic bacteria and some aerobic opportunists were infrequent in urine. Semen is a suitable specimen for the diagnosis of prostatitis.     

Sporadic aneuploidy in PHA-stimulated lymphocytes of trisomies 21, 18, and 13

Following the observation detected in a previous study that X chromosome monosomy in Turner's syndrome genotypes was associated with a sporadic loss and/or gain of other chromosomes, we studied here whether this instability is a consistent finding in constitutional autosomal trisomies. We used PHA-stimulated lymphocytes derived from 14 patients (10 patients with trisomy 21, 2 with trisomy 18, and 2 with trisomy 13). Fourteen healthy controls were compared. Fluorescence in situ hybridization, applied at interphase cells, was used to evaluate the level of aneuploidy for 3 randomly selected chromosomes (autosomes 8, 15, and 16) in each sample. For each tested chromosome, our results showed a significantly higher level of aneuploid cells in the samples from the patients than in those from controls, with no difference between the patient groups. The mean level of aneuploid cells (percentage) for all 3 tested autosomes was almost twice as high in the patient samples as in the control samples. The aneuploidy level was mainly due to monosomy, which was significantly higher in the samples from the patients than in those from controls for each one of the tested chromosomes, with no difference between the patient groups. The mean level of monosomic cells (percentage) for all 3 tested chromosomes was almost twice as high in the patient samples as in the control samples. Our study shows that various constitutional autosomal trisomies are associated with an increased frequency of non-chromosome specific aneuploidy and is a continuation of the previous study documenting sporadic aneuploidy in Turner's sample cells. It is possible that primary aneuploid cells destabilize their own genome resulting in variable aneuploidy of other chromosomes. It is also possible that one or several common factor(s) is/are involved in both constitutional and sporadic aneuploidy.