Merzenich et al, BMC Public Health 2010, 10:24 http://www.biomedcentral.com/1471-2458/10/24 concentration described in the considered studies varies between 37 × 106/ml and 159 × 106/ml ejaculate fluid. The lowest mean sperm density was found in a study con- ducted in Brazil among 501 men [4]. The highest mean sperm concentration is derived from an Irish study which of only 10 men [5]. The sample size of the considered studies shows a wide range from 7 [6] to 4435 included men [7]. The central message of the original meta-analysis, that sperm density had decreased globally by about 50% over the past 50 to 60 years, attracted considerable attention and generated much controversy, as well. However, this conclusion should to be interpreted with caution. Rea- sons for heterogeneity between studies like geographical and ethnical variation, different study designs and differ- ent methodological standards have to be considered, as well as selection bias and measurement bias. Important methodological pitfalls in fertility studies will be dis- cussed. Discussion Geographical and time-related variation Fisch and co-workers determined whether geographic variations in sperm count might bias the conclusions drawn from studies of semen quality. Briefly, of the 61 studies in the meta-analysis of Carlsen et al. [1] only 20 included more than 100 individuals. A reanalysis of these 20 studies revealed that the majority of studies published before 1970 were from USA, mainly New York. These studies represented locations with the highest sperm counts. In contrast, after 1970, most of the studies were from locations not included earlier. Selection bias due to geographical and ethnic variations could account for the observed decline in sperm quality [8]. However, when the subgroup of studies from the USA (28 studies, including data on 8.329 men) was analyzed separately, a similar trend of decreasing mean sperm values was observed [1]. On the other hand, Lipshultz argued that when all the larger studies containing only New York data are excluded from the meta-analysis, a second linear regres- sion analysis detects no decline in sperm density [9]. The sperm count issue has been extensively investi- gated since 1992 and the decrease is supported by addi- tional studies. For example, it was reported that sperm counts of 1351 fertile men (semen donors) in Paris decreased by 2.1 percent per year from 89 × 106/ml in 1973 to 60× 106/ml in 1992 (p < 0.001) [10]. Sperm motil- ity also decreased in these individuals. The mean seminal volume was 3.8 ml and did not change during the period from 1973 to 1992. Irvine et al. also examined sperm quality in 571 semen donors in Scotland by birth cohort groups [11]. They reported a significant decrease in median sperm concentration, total number of sperm in the ejaculate and total number of motile sperm in donors Page 2 of 5 born between 1970 and 1974 compared to men before 1959. On the other hand, several studies confirm that in some locations sperm counts have not decreased over the past 20 to 25 years. More importantly, the results clearly show a remarkable variability in sperm counts at different geo- graphical locations. The mean sperm counts in 302 men in the Toulouse area were unchanged over the period from 1977 to 1992 [12] and their mean sperm count (83 × 106 ml) was significantly higher than observed in the Paris study [10]. Furthermore, it was reported that the highest sperm counts recorded in Finland were found in men from rural areas accompanied by a low incidence of tes- ticular cancer [13]. These findings suggest that urban life- style or environmental factors might be an important etiological factor of testicular malfunction and disease. Fisch et al. [14] conducted a study comprising 1,283 men who banked sperm before vasectomy in three differ- ent sperm banks in the United States from 1970-1994. A slight but significant increase in mean sperm concentra- tion from 77 × 106 ml to 89 × 106 ml over the past 25-year period was found. Furthermore, marked differences in semen characteristics between the New York, Minnesota, and California sperm banks were found. Sperm concen- tration and motility was highest in New York and lowest in California. It was argued that the data set analyzed by Carlsen et al. [1] was not equally distributed between the decades: 79% of the publications and 88% of the volunteers of all studies were clustered between 1970 and 1990. If joint-analyses were conducted with studies from this period only, an increase in sperm concentrations would become evident. Thus, only 21% of the studies and 12% of the volunteers analyzed before 1970 have caused the regression to have a statistically negative slope [15,16]. Looking at sperm count studies which have been pub- lished since 1992, it becomes evident that over the past 20 years, sperm quality has decreased in some but not all locations. The results also show that sperm counts can vary widely between and within countries. These major geographical differences suggest that the results of the former meta-analyses of sperm count may be biased by geographical confounding. Selection bias The WHO recommends that in order to determine nor- mal ranges of semen quality "specimens should be evalu- ated from men who have recently achieved a pregnancy, preferably within 12 months of the couple ceasing contra- ception" [17]. In the former meta-analyses [1-3] a precise definition of "proven fertility" is not given. Nevertheless, a significant decline in mean sperm concentration was seen when the 39 papers reporting data on men with "proven fertility" where analyzed separately. In the remaining 22 publications, men were unselected with respect to fertil-