A cohort effect on serum testosterone levels in Finnish men.

OBJECTIVE: To investigate whether a population-level decline in serum testosterone exists in Finnish men. In comparison with other European populations, Finnish men have compared well in the studies of reproductive health (i.e. semen quality, incidence of cryptorchidism and testicular cancer); thus, we expected no significant cohort-dependent decrease in serum testosterone. METHODS: We analysed serum levels of testosterone, gonadotrophin and sex hormone binding globulin (SHBG) in 3271 men representing different ages (25-74 years) and birth cohorts within three large Finnish population surveys conducted in 1972, 1977 and 2002. RESULTS: Serum testosterone levels decreased (from 25.3 nmol/l in 25- to 29-year-old men gradually to 16.9 nmol/l in 70- to 74-year-old men), whereas SHBG and gonadotrophin levels increased with increasing age. In addition, a significant secular trend in testosterone (total and free), SHBG and gonadotrophin levels was observed with lower levels in more recently born age-matched men. Serum testosterone level decreased in men aged 60-69 years from 21.9 nmol/l (men born 1913-1922) to 13.8 nmol/l (men born 1942-1951). These decreases remained significant following adjustment for BMI. An age-independent birth cohort effect existed on reproductive hormones measured in the Finnish men. In concert with the lower free testosterone levels, we observed lower gonadotrophin levels, suggesting that while there may be detrimental changes at the gonad level, the hypothalamus-pituitary-axis is not responding appropriately to this change. CONCLUSIONS: The more recently born Finnish men have lower testosterone levels than their earlier born peers. This study offers no explanation for this substantial recent adverse development.

Semen quality and reproductive hormone levels in men from Southern Spain.

In North European countries, a significant difference in semen quality among young men has been shown. Men from the western countries, Denmark, Germany and Norway, have lower semen quality than men from the eastern countries Finland, Estonia and Lithuania. Similarly, men in the western countries have a higher risk of testicular cancer. According to the testicular dysgenesis syndrome (TDS) concept that suggests a link between risk of impaired semen quality and increased risk of testicular cancer, Spanish men would be expected to have a semen quality at a normal level because of their very low testis cancer risk. We therefore investigated 273 men from the Almeria region in the Southern Spain to test this hypothesis. The men delivered semen samples, underwent physical examinations, had a blood sample drawn and provided information on lifestyle and reproductive health parameters. The investigations took place from November 2001 to December 2002. Adjusting for effects of confounders, the median sperm concentration and total sperm count were 62 (95% confidence interval 47-82) million/mL and 206 (153-278) million, respectively. The median numbers of motile and morphologically normal spermatozoa assessed according to strict criteria were 59% (57-62%) and 9.4% (8.6-10.0%), respectively. The median total testosterone and calculated free androgen index were 28 nm (26-30) and 95 (88-103), respectively. Assuming that the investigated men, to a large extent, are representative of the population of young men the Southern Spain, the results show that these have normal semen quality and reproductive hormone levels as expected in a population with a low incidence of testicular cancer.

Recent adverse trends in semen quality and testis cancer incidence among Finnish men.

Impaired semen quality and testicular cancer may be linked through a testicular dysgenesis syndrome of foetal origin. The incidence of testis cancer has been shown to increase among Finnish men, whereas there is no recent publication describing temporal trends in semen quality. Therefore, we carried out a prospective semen quality study and a registry study of testis cancer incidence among Finnish men to explore recent trends. A total of 858 men were investigated in the semen quality study during 1998-2006. Median sperm concentrations were 67 (95% CI 57-80) million/mL, 60 (51-71) and 48 (39-60) for birth cohorts 1979-81, 1982-83 and 1987; total sperm counts 227 (189-272) million, 202 (170-240) and 165 (132-207); total number of morphologically normal spermatozoa 18 (14-23) million, 15 (12-19) and 11 (8-15). Men aged 10-59 years at the time of diagnosis with testicular cancer during 1954-2008 were included in the registry study, which confirmed the increasing incidence of testicular cancer in recent cohorts. These simultaneous and rapidly occurring adverse trends suggest that the underlying causes are environmental and, as such, preventable. Our findings necessitate not only further surveillance of male reproductive health but also research to detect and remove the underlying factors.

Time to pregnancy and semen parameters: a cross-sectional study among fertile couples from four European cities.

BACKGROUND: In fertile populations, little is known about the association between semen parameters and time to pregnancy (TTP). METHODS: Pregnant women from Copenhagen, Edinburgh, Paris and Turku who conceived without medical intervention were asked for their TTP (942 couples), and their partners provided a semen sample. The proportion of morphologically normal sperm and the multiple anomalies index (MAI, ratio of the total number of anomalies to the number of abnormal sperm) were centrally estimated. We estimated rate ratios for the occurrence of a pregnancy by a discrete survival model, adjusted for sexual activity and female factors affecting fecundity. RESULTS: Increasing sperm concentration influenced TTP up to 55 x 10(6)/ml. The proportion of morphologically normal sperm influenced TTP up to 39% according to David's criteria, and this association held among the subjects with a sperm concentration >55 x 10(6)/ml. For strict criteria, the threshold value was 19% normal sperm. An increase of 0.5 in MAI was associated with an adjusted rate ratio for the occurrence of a pregnancy of 0.68 (95% confidence interval: 0.54-0.85). CONCLUSIONS: These results highlight the importance of sperm morphology parameters and indicate that the effect of proportion of normal sperm on TTP may be independent of sperm concentration.

Regional differences in waiting time to pregnancy among fertile couples from four European cities.

BACKGROUND: A previous European study found a longer time to pregnancy (TTP) among fertile women from Paris compared with women from other Western European countries. A co-ordinated, cross-sectional study of pregnant couples from Denmark (Copenhagen), France (Paris), Scotland (Edinburgh) and Finland (Turku) was therefore undertaken to assess differences in waiting TTP among couples from these cities. METHODS: Pregnant women were invited to participate when they showed up for their first antenatal visit in one of the four centres. Inclusion criteria included that their partner was 20-45 years of age and born in the country in which he was currently living and that the pregnancy was achieved without fertility treatment. Both partners filled in a questionnaire and the man underwent a physical examination and delivered a semen sample (Turku: n = 237, Copenhagen: n = 302, Edinburgh: n = 212, Paris: n = 191). RESULTS: French couples had a decreased probability of conception compared with couples from the other three countries, although only after adjustment for confounders. No significant differences between couples from the three other countries were found. CONCLUSION: The observed geographical differences in TTP remain unexplained and were not due to differences in semen quality, but may be caused by varying exposures to environmental factors or psychological distress. In addition, selection bias due to the low participation rates cannot be ruled out. Future studies examining the causes for geographical differences in TTP are needed.

Sperm morphological defects related to environment, lifestyle and medical history of 1001 male partners of pregnant women from four European cities.

BACKGROUND: Recently, differences in semen quality have been found among the partners of pregnant women from four European cities: Turku, Copenhagen, Edinburgh and Paris. METHODS: During this study, slides from the four centres were subjected to a centralized assessment of sperm morphology. The percentages of sperm defects were recorded using a multiple-entry classification enabling the calculation of the multiple anomalies index (MAI), which is the mean number of anomalies per abnormal sperm. The relationships between various sperm abnormalities and self-reported data on medical history, lifestyle and occupational factors were examined. RESULTS: Significant differences in the MAI and most of the sperm defects were found between the four cities, and more abnormalities were found in spring than in winter. An increase in some sperm abnormalities was related to medical treatment of the mother during pregnancy, higher birthweight and previous treatment for cryptorchidism. Significant variations of several sperm defects were related to stress, weekly working time, occupational posture and metal welding, suggesting directions for further exposure studies. CONCLUSION: The present study indicated that the detailed assessment of sperm abnormalities is a useful biomarker of the effect of various external factors which may qualitatively affect human spermatogenesis.

Regional differences in semen quality in Europe.

Recent reports have indicated a decrease in semen quality of men in some countries, and suggested regional differences. A study was undertaken of semen samples from 1082 fertile men from four European cities (Copenhagen, Denmark; Paris, France; Edinburgh, Scotland; and Turku, Finland). Semen analysis was standardized, inter-laboratory differences in assessment of sperm concentration were evaluated, and morphology assessment centralized. Lowest sperm concentrations and total counts were detected for Danish men, followed by French and Scottish men. Finnish men had the highest sperm counts. Men from Edinburgh had the highest proportion of motile spermatozoa, followed by men from Turku, Copenhagen and Paris. Only the differences between Paris/Edinburgh and Paris/Turku were statistically significant (P < 0.003 and P < 0.002 respectively). No significant differences in morphology were detected. A general seasonal variation in sperm concentration (summer 70% of winter) and total sperm count (summer 72% of winter) was detected. Semen quality of a 'standardized' man (30 years old, fertile, ejaculation abstinence of 96 h) were estimated. Typically, sperm concentrations (x 10(6)/ml) for winter/summer were: Turku 132/93; Edinburgh 119/84; Paris 103/73; and Copenhagen 98/69. These differences in semen quality may indicate different environmental exposures or lifestyle changes in the four populations. However, it remains to be seen whether such changes can account for these differences. These data may also serve as a reference point for future studies on time trends in semen quality in Europe.

Reassessment of sperm morphology of archival semen smears from the period 1980--1994.

Several reports have suggested that sperm counts of normal men have declined in many geographical regions during the last decades. Deterioration of sperm morphology has also been reported in some studies covering long sample collecting periods. The original semen analysis data of our semen laboratory from the period 1980--1994 showed a significant decline in the proportion of spermatozoa with normal morphology. The finding was, however, questioned because of changes in sperm morphology assessment criteria during the study period. In the present study 1745 smears were re-analysed to cover evenly the whole study period. The samples were examined in random order by using strict assessment criteria. Multiple linear regression analysis of the re-analysed data showed no effect of the year of sample delivery on sperm morphology between the years 1980 and 1994. However, there was a significant decline in the proportion of normal spermatozoa with later year of men's birth.

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis.

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.

Semen quality among Danish and Finnish men attempting to conceive. The Danish First Pregnancy Planner Study Team.

OBJECTIVE: To assess differences in semen quality between similar populations from Denmark and Finland. DESIGN: Comparison of semen quality between 221 Finnish men (of whom 115 had no proven fertility) and 411 Danish men with no proven fertility in two follow-up studies among normal couples trying to conceive. METHODS: In Finland male partners of couples without experienced infertility attempting to conceive were recruited through advertisements in local newspapers from 1984 to 1986. From 1992 to 1995 Danish men who lived with a partner and who had not attempted to achieve a pregnancy previously were recruited through their union when they discontinued birth control. All semen analyses were performed in accordance with the World Health Organization guidelines. RESULTS: Median sperm concentration, total sperm count and the percentage of morphologically normal spermatozoa were significantly higher among the Finnish men without proven fertility (104.0 million/ml, 304.0 million and 58% respectively) compared with the Danish men (53.0 million/ml, 140.8 million, and 41% respectively). Sperm concentration was 105.7% (95% confidence interval (CI) 58.1%-167.6%) and total sperm count was 127.4% (95% CI 71.4%-201.6%) higher among Finnish men without proven fertility than among Danish men after control for confounders. CONCLUSIONS: Some, but hardly all, of the observed difference in semen quality may be explained by differences in recruitment procedures, selection of the men and by methodological differences in semen analysis between the two countries. Also a birth cohort effect may explain some of the differences between countries as the Finnish men were recruited 11 years before the Danish men. Therefore, follow-up studies with identical recruitment and selection of men from the two countries are needed.

Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for nonendothelial targets for VEGF.

Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.

Probable spermatozoal diploidy in the semen of a golden retriever.

The semen of a 3-year-old golden retriever was examined for breeding purposes. When the morphology of the spermatozoa was analysed for the first time, 37% were observed to have giant heads. In most of the giant heads, a diadem defect was also found. The dog was successfully used for breeding. On re-examination, the percentage of giant heads was found to be greater than before. The right testicle exhibited tissue softening. To determine the reason for the defect, an aspiration needle biopsy was performed and ultrasound examination undertaken. In the biopsy smears, both normal spermatozoa and spermatozoa with giant heads were found. On ultrasonography, the echogenicities of both testicles were the same, and normal. DNA flow cytometry was performed to determine the DNA content of the spermatozoa. Two populations of sperm cells were detected, one having a median fluorescent intensity twice as high as that of normal spermatozoa, suggesting a diploid DNA content. Transmission electron microscopy (TEM) was performed to find out whether the altered intensity correlated with the ultrastructure of the spermatozoa. The nuclei of the sperm heads showed a normal chromatin condensation. Semen quality became worse over a period of 2 years, with 60% giant heads in the last sample. The process was considered to be progressive spermatogenic degeneration with diploidy. Relatives examined did not suggest any hereditary predisposition to the problem. The male was still fertile at the time of the last sample collected and sired a litter of 10 healthy puppies.

Morphometry of normal and teratozoospermic canine sperm heads using an image analyzer: work in progress.

Combining the traditional morphologic evaluation of spermatozoa with computer assisted image analysis adds randomness, objectivity, repeatability and accuracy to morphometric measurements. We collected semen from 10 fertile, normospermic dogs aged 1 to 7 yr and from 3 teratozoospermic breed-matched dogs. Sperm head morphology was examined in Giemsa-stained smears by light microscopy, using a computer-assisted image analyzer and by transmission electron microscopy. We found significant variation in sperm head area, length, width and degree of roundness among normospermic individual dogs, indicating that it would be necessary to examine many more dogs before the size and shape of normal dog spermatozoa could be determined. The normospermic dogs were used as controls for the teratozoospermic cases. Case 1: A 2-yr-old subfertile Cavalier King Charles Spaniel had semen with small and narrow-based sperm heads and a proximal cytoplasmic droplet in most of the spermatozoa. With the image analysis system, sperm heads were shown to be smaller and more oval than in normospermic dogs. The variatons in size and shape were similar in magnitude to those of control dogs. An examined infertile half-brother had similar semen quality. Case 2: A 3-yr-old Petit Basset Griffon Vendeen with 2 unsuccesfull matings exhibited spermatozoa with severe abnormalities. Measured by image analyzer, sperm heads were irregular in shape and very small in area. One of the two littermates examined had semen of the same quality as the case dog. Case 3: A 3-yr-old fertile Golden Retriever had semen with giant sperm heads in about 50% of spermatozoa. Image analyzing results revealed 2 populations of different sized sperm heads. Giant heads consisted of 52.2% of all spermatozoa. The results of the study reported here suggest that the image analysis technique may be useful in evaluating structural changes in sperm morphology, supplementing visual assessment that is used in conventional methods.

High and unchanged sperm counts of Finnish men.

Several recent reports have suggested that the sperm counts of normal men are declining in most countries. In this study the sperm counts of Finnish men, and their possible changes during the past 28 years, were investigated. The material consisted of semen samples from 238 normal healthy men of unknown fertility and 5481 men from infertile couples. The means (medians) of semen volume, sperm density and total sperm count in normal men were 3.3 (3.0) ml, 133.9 (94.0) x 10(6)/ml and 396.6 (309.0) x 10(6), respectively. These parameters and the relative frequency distribution of the sperm density were similar to those reported elsewhere in the 1940s. Multiple linear regression analysis revealed a significant decrease in semen volume, whereas sperm density and total sperm count of infertile men had not changed significantly during the past 28 years. In addition, no change in sperm counts was associated with the year of men's birth.

Electron microscopic immunolocalization of the 18 and 29 kilodalton secretory proteins in the mouse epididymis: evidence for differential uptake by clear cells.

In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747-766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron-lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis. Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda. These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different.

MIP-1 alpha is a regulator of mitotic and meiotic DNA synthesis during spermatogenesis.

To find out whether macrophage inflammatory protein-1 alpha (MIP-1 alpha) has a role in the regulation of germ cell development, we studied its effects on spermatogenic stage-specific DNA synthesis in vitro. MIP-1 alpha increased the DNA synthesis of primitive type A2-4 spermatogonia and of premeiotic cells, whereas the DNA synthesis of more differentiated intermediate and type B spermatogonia was inhibited when cultured in the presence of MIP-1 alpha. An antibody against MIP-1 alpha cross-reacted with a protein of 15 kDa from every spermatogenic stage of rat seminiferous epithelium. Immunohistochemical studies with the same antibody revealed a complex pattern of MIP-1 alpha localization both in primitive and advanced spermatogenic cells. These observations suggest that MIP-1 alpha is a local regulator of mitotic and meiotic DNA synthesis.

Isolation and characterization of a 25-kilodalton protein from mouse testis: sequence homology with a phospholipid-binding protein.

A 25-kDa epididymal secretory protein (MEP 9), isolated from mouse epididymal fluid, has recently been characterized in our laboratory [Rankin et al., Biol Reprod 1992; 46:747-766]. The polyclonal antibody raised against this protein was found to recognize a 25-kDa component in epididymal fluid and testicular extract. The 25-kDa testicular antigen (MTP) was purified by means of ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography; MTP was found to be similar to MEP 9 in several properties including molecular mass (25 kDa), isoelectric point (pI 6.0), and immunoreactivity when the proteins were resolved in the presence of SDS (one-dimensional and two-dimensional PAGE). However, when the proteins were resolved under non-denaturing conditions, MTP showed strong immunoreactivity while MEP 9 did not. This observation suggests that although the 25-kDa antigens from the epididymal fluid and testicular extract are quite similar, they may have different immunological conformations. When analyzed for amino acid composition and partial amino acid sequence, the testicular antigen showed substantial homology (> 80%) with a phosphatidylethanolamine-binding protein characterized from bovine brain. MTP also showed phosphatidylethanolamine-binding activity (Kd = 1.95 x 10(-5) M, Bmax = 1.86 nmol/micrograms MTP), suggesting that the mouse 25-kDa protein is a member of the phospholipid-binding protein family and may have a role in lipid metabolism during sperm maturation.

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.