Artificial intelligence in the fertility clinic: status, pitfalls and possibilities.

In recent years, the amount of data produced in the field of ART has increased exponentially. The diversity of data is large, ranging from videos to tabular data. At the same time, artificial intelligence (AI) is progressively used in medical practice and may become a promising tool to improve success rates with ART. AI models may compensate for the lack of objectivity in several critical procedures in fertility clinics, especially embryo and sperm assessments. Various models have been developed, and even though several of them show promising performance, there are still many challenges to overcome. In this review, we present recent research on AI in the context of ART. We discuss the strengths and weaknesses of the presented methods, especially regarding clinical relevance. We also address the pitfalls hampering successful use of AI in the clinic and discuss future possibilities and important aspects to make AI truly useful for ART.

Anti-Müllerian hormone in seminal plasma and serum: association with sperm count and sperm motility.

STUDY QUESTION: Is anti-Müllerian hormone (AMH) in seminal plasma and serum associated with sperm count and sperm motility? SUMMARY ANSWER: AMH in seminal plasma is positively associated with sperm concentration, total sperm count, and progressive sperm motility, while no association was found between serum AMH levels and semen characteristics. WHAT IS KNOWN ALREADY: AMH is secreted by the Sertoli cells and is detectable in both serum and seminal plasma in adult men. It has been suggested as a marker of spermatogenesis, however, its function in the adult male is largely unknown. STUDY DESIGN, SIZE, DURATION: Participants were recruited in between 2008 and 2013, from the general population (n = 94) and from couples with female factor infertility in a fertility clinic (n = 32). AMH data were available for 126 participants. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mean age of the participants was 36 years, and BMI was between 19 and 39 kg/m(2). Semen quality was evaluated by semen analysis according to the World Health Organization, and AMH levels were measured in seminal plasma. Blood samples were analyzed for AMH, total testosterone, FSH, LH, and inhibin B. AMH analysis was performed using the improved Beckman Coulter method. MAIN RESULTS AND THE ROLE OF CHANCE: The central 95% intervals of AMH concentrations were 2-2812 pmol/l in seminal plasma and 15-134 pmol/l in serum. Total AMH (pmol/ejaculate) in seminal plasma was positively associated with sperm concentration (B = 0.177, P< 0.001) and total sperm count (B = 0.212, P< 0.001) when adjusted for age, BMI, time of abstinence, and positively associated with progressive sperm motility (B = 6.762, P = 0.001) when adjusted for age, BMI, time of abstinence, and site of sample collection. No association was found between serum AMH and semen characteristics. Serum levels of inhibin B were positively correlated with total AMH in seminal plasma (B = 18.52, P< 0.001) and concentration of AMH in serum (B = 0.507, P< 0.001). LIMITATIONS, REASONS FOR CAUTION: Participants were recruited both from the general population and from a fertility clinic. This may limit the applicability to men in the general population. WIDER IMPLICATIONS OF THE FINDINGS: The AMH levels found in this study show large inter-individual variation, especially in seminal plasma. AMH in seminal plasma may serve as a marker of sperm production, however, in the lower range the predictive value is low. STUDY FUNDING/COMPETING INTERESTS: All funding for this study was received from Oslo and Akershus University College of Applied Sciences. The authors have no conflicts of interest to declare.

Profiling of the small RNA populations in human testicular germ cell tumors shows global loss of piRNAs.

BACKGROUND: Small non-coding RNAs play essential roles in gene regulation, however, the interplay between RNA groups, their expression levels and deregulations in tumorigenesis requires additional exploration. In particular, a comprehensive analysis of microRNA (miRNA), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs in human testis and testicular germ cell tumor (TGCT) is lacking. RESULTS: We performed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma in situ, and 12 normal testis samples. miRNA was the most common group among the sequences 18-24 nt in length and showed histology-specific expression. In normal samples, most sequences 25-31 nucleotides in length displayed piRNA characteristics, whereas a large proportion of the sequences 32-36 nt length was derived from tRNAs. Expression analyses of the piRNA population demonstrated global loss in all TGCT subtypes compared to normal testis. In addition, three 5' small tRNA fragments and 23 miRNAs showed significant (p < 10(-6)) differential expression in cancer vs normal samples. CONCLUSIONS: We have documented significant changes in the small RNA populations in normal adult testicular tissue and TGCT samples. Although components of the same pathways might be involved in miRNA, piRNA and tRNA-derived small RNA biogenesis, our results showed that the response to the carcinogenic process differs between these pathways, suggesting independent regulation of their biogenesis. Overall, the small RNA deregulation in TGCT provides new insight into the small RNA interplay.

No association between body mass index and sperm DNA integrity.

STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including mainly subfertile men, have shown conflicting results regarding the influence of overweight and obesity on sperm DNA integrity. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was based on semen samples from 1503 men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included two cohorts (cohort A and B) of military recruits (n = 275, n = 304, respectively), one group (cohort C) of fertile men and men without known fertility problems (n = 724), and one group (cohort D) of men between 19 and 40 years without known fertility problems (n = 200). In all cohorts, data were available on BMI, DFI as measured by the sperm chromatin structure assay (SCSA), standard semen characteristics, and potential confounders (age, abstinence time, smoking habits). The subjects were categorized according to BMI into four groups: underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)), overweight (25.0-29.9 kg/m(2)) and obese (≥30.0 kg/m(2)). Using a linear regression model, the inter-group differences in DFI were calculated. Furthermore with the normal-weight group as the reference, the odds ratios (ORs) for DFI > 20% and DFI > 30%, were calculated for the other groups. Calculations were made for the material as a whole and after exclusion of cohort C which included proven fertile men. MAIN RESULTS AND THE ROLE OF CHANCE: We found that normal-weight men had significantly higher DFI than overweight men, with a mean difference of 1.13% (95% CI: 1.05-1.22%); P = 0.001). Overweight men had a reduced risk of having DFI ≥ 20% and DFI ≥ 30%, compared with normal-weight men; adjusted odds ratio (OR) = 0.61 (95% CI: 0.42-0.88; P < 0.01) and adjusted OR = 0.48 (95% CI: 0.28-0.84; P < 0.01), respectively. When excluding cohort C, the statistical significance was lost. Regarding standard semen parameters, we found that obese men had a higher percentage of progressive motile spermatozoa than normal-weight men; mean difference 1.15% (95% CI: 1.02-1.30%, P < 0.05) but the significance was lost when excluding cohort C. All other standard semen parameters were unaffected by BMI. LIMITATIONS, REASONS FOR CAUTION: A main limitation might be the cross-sectional nature of the data. Furthermore our study included a significant proportion of men with proven fertility (75% of cohort C, n = 550), and could therefore be biased toward fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our study indicates that overweight per se is not associated with a higher level of sperm DNA damage. STUDY FUNDING/COMPETING INTERESTS: This research has been given grants from the following: EU 5th and 7th framework program (Inuendo and Clear projects, [Contracts no. QLK4-CT-2001-00202 and FP7-ENV-2008-1-226217)]), the Swedish Research Council (Grants No. 2007-2590, 521-2004-6072 and 521-2002-3907); the Swedish Governmental Funding for Clinical Research, Skåne county council's research and development foundation, MAS Funds, University Hospital MAS Foundation in Malmö, Crafoordska Fund, Ove Tulefjords Fund, Foundation for Urological Research, Fundacion Federico SA, and Gunnar Nilssons Cancer Fund. The authors declare that there are no conflicts of interest.

Genetic variations associated with the effect of testicular cancer treatment on gonadal hormones.

STUDY QUESTION: Do genetic variations in the testosterone pathway genes modify the effect of treatment on the levels of testosterone and LH in long-term testicular cancer (TC) survivors (TCSs)? SUMMARY ANSWER: Variations in LH receptor (LHR) and in 5α-reductase II (SRD5A2) genes may modify the effect of TC treatment on testosterone levels, whereas genetic variations in the androgen receptor (AR) may modify the effect on LH levels. WHAT IS KNOWN ALREADY: TCSs experience variable degrees of long-term reduction in gonadal function after treatment. This variability can in part be explained by treatment intensity, but may also be due to individual variations in genes involved in the function and metabolism of reproductive hormones. STUDY DESIGN, SIZE, DURATION: Cross-sectional study on testosterone and LH levels in 637 Norwegian TCSs in relation to genetic variants and TC treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: The single nucleotide polymorphisms LHR Asn291Ser (rs12470652) and Ser312Asn (rs2293275), as well as SRD5A2 Ala49Thr (rs9282858) and Val89Leu (rs523349) were analyzed by allele-specific PCR. The insertion polymorphism LHR InsLQ (rs4539842) was analyzed by sequencing. The numbers of AR CAG and GGN repeats were determined by capillary electrophoresis. Blood samples were collected 5-21 years after diagnosis (median 11 years) and serum total testosterone and LH were analyzed by commercial immunoassays. The TCSs were divided into four groups according to their treatment; surgery only, radiotherapy and chemotherapy with ≤850 or >850 mg of cisplatin. Polymorphisms presenting P < 0.1 for the interaction term with treatment in an initial two-way analysis of covariance (ANCOVA) were investigated further in two consecutive one-way ANCOVA analyses to elucidate the interaction between treatment and genotype. MAIN RESULTS AND THE ROLE OF CHANCE: For the whole group of TCSs, there were no significant differences between the hormone levels in homozygotes for the wild type and carriers of at least one polymorphic allele for the investigated polymorphisms. Three of the polymorphisms showed signs of interaction with treatment, i.e. LHR InsLQ, SRD5A2 A49T and the AR CAG repeat. Follow-up analyses revealed three situations where only one of the genotypes of the polymorphism where associated with significantly different hormone levels after surgery compared with after additional cytotoxic treatment: For LHR InsLQ, only the wild-type allele was associated with lower testosterone levels after cisplatin > 850 mg compared with after surgery (24% lower, P < 0.001). For SRD5A2 A49T, testosterone levels were lower after radiotherapy compared with after surgery, but only for the heterozygotes for the polymorphism (39% lower, P = 0.001). In comparison, the testosterone levels were just slightly lower after radiotherapy (6% lower, P = 0.039) or cisplatin ≤ 850 mg (7% lower, P = 0.041), compared with surgery, independent of genotypes. For AR CAG, only the reference length of CAG = 21-22 had significantly higher LH levels after cisplatin ≤ 850 mg compared with after surgery (70% higher, P < 0.001). Independent of genotypes, however, LH levels after cisplatin ≤ 850 mg were only 26% higher than after surgery (P = 0.005). LIMITATIONS, REASONS FOR CAUTION: Unadjusted P-values are presented. For analysis involving genotypes, the level of statistical significance was adjusted for the total number of polymorphisms tested, n = 7, i.e. to P < 0.007 (0.5/7). The rather weak associations indicate that additional polymorphisms are involved in the modulation. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study supporting the notion that polymorphisms may explain at least some of the inter-individual differences in endocrine response to TC treatment. Our findings suggest that individuals with certain genotypes may be more vulnerable to certain treatments. Knowledge on genetic predisposition concerning treatment-related endocrine gonadotoxicity to different treatment regimens may help tailoring TC therapy when possible. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Research Council of Norway (Grant No. 160619). There were no competing interests.

Genetic variation in AKT1, PTEN and the 8q24 locus, and the risk of testicular germ cell tumor.

STUDY QUESTION: Is there an association between testicular germ cell tumor (TGCT) and genetic polymorphisms in AKT1, PTEN and the 8q24 locus? SUMMARY ANSWER: Our findings suggest that genetic variation in PTEN may influence the risk of TGCT. WHAT IS KNOWN ALREADY: There is strong evidence that genetic variation influences the risk of TGCT. The oncogene, AKT1, the tumor suppressor gene, PTEN and the chromosome 8q24 locus play important roles in cancer development in general. STUDY DESIGN, SIZE, DURATION: We have conducted a population-based Norwegian-Swedish case-parent study, based on cases diagnosed in 1990-2008, including 831 triads (TGCT case and both parents), 474 dyads (TGCT case and one parent) and 712 singletons (only the TGCT case). In addition we expanded the study to include 3922 unrelated male controls from the TwinGene project. PARTICIPANTS/MATERIALS, SETTING, METHODS: We genotyped 26 single nucleotide polymorphisms (SNPs) in AKT1, PTEN and the 8q24 locus. First, triads and dyads were included in a likelihood-based association test. To increase the statistical power, case singletons and controls from the TwinGene project were included in a single test for association. We examined if the allelic effect on TGCT risk differed by histological subgroup, country of origin or parent of origin. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated with Bonferroni correction (P bonf) for multiple testing. MAIN RESULTS AND THE ROLE OF CHANCE: In the case-parent analyses, none of the 26 SNPs were significantly associated with TGCT. Of the 23 SNPs investigated in the combined study, one SNP in PTEN (rs11202586) remained associated with TGCT risk after adjusting for multiple testing (OR = 1.16, 95% CI = 1.06-1.28, P bonf = 0.040). We found no difference in risk according to histological subgroup, parent of origin or between countries. LIMITATIONS, REASONS FOR CAUTION: Our study is strengthened by the population-based design and large sample size, which gives high power to detect risk alleles. The reported association was not highly significant, and although it was based on an a priori hypothesis of this tumor suppressor gene being implicated in the etiology of TGCT, replication studies, as well as functional studies of this polymorphism, are warranted. WIDER IMPLICATIONS OF THE FINDINGS: We report, to our knowledge, a novel association between TGCT and a marker in the tumor suppressor gene PTEN. Previous studies have linked PTEN to TGCT etiology, and there is also a link between PTEN and KITLG, which contains TGCT susceptibility loci revealed through recent genome-wide studies.

Gene variations in sex hormone pathways and the risk of testicular germ cell tumour: a case-parent triad study in a Norwegian-Swedish population.

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common cancer in young men, and an imbalance between the estrogen and androgen levels in utero is hypothesized to influence TGCT risk. Thus, polymorphisms in genes involved in the action of sex hormones may contribute to variability in an individual's susceptibility to TGCT. METHODS: We conducted a Norwegian-Swedish case-parent study. A total of 105 single-nucleotide polymorphisms (SNPs) in 20 sex hormone pathway genes were genotyped using Sequenom MassArray iPLEX Gold, in 831 complete triads and 474 dyads. To increase the statistical power, the analysis was expanded to include 712 case singletons and 3922 Swedish controls, thus including triads, dyads and the case-control samples in a single test for association. Analysis for allelic associations was performed with the UNPHASED program, using a likelihood-based association test for nuclear families with missing data, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. False discovery rate (FDR) was used to adjust for multiple testing. RESULTS: Five genetic variants across the ESR2 gene [encoding estrogen receptor beta (ERß)] were statistically significantly associated with the risk of TGCT. In the case-parent analysis, the markers rs12434245 and rs10137185 were associated with a reduced risk of TGCT (OR = 0.66 and 0.72, respectively; both FDRs <5%), whereas rs2978381 and rs12435857 were associated with an increased risk of TGCT (OR = 1.21 and 1.19, respectively; both FDRs <5%). In the combined case-parent/case-control analysis, rs12435857 and rs10146204 were associated with an increased risk of TGCT (OR = 1.15 and 1.13, respectively; both FDRs <5%), whereas rs10137185 was associated with a reduced risk of TGCT (OR = 0.79, FDR <5%). In addition, we found that three genetic variants in CYP19A1 (encoding aromatase) were statistically significantly associated with the risk of TGCT in the case-parent analysis. The T alleles of the rs2414099, rs8025374 and rs3751592 SNPs were associated with an increased risk of TGCT (OR = 1.30, 1.30 and 1.21, respectively; all FDRs <5%). We found no statistically significant differences in allelic effect estimates between parental inherited genetic variation in the sex hormone pathways and TGCT risk in the offspring, and no evidence of heterogeneity between seminomas and non-seminomas, or between the Norwegian and the Swedish population, in any of the SNPs examined. CONCLUSIONS: Our findings provide support for ERß and aromatase being implicated in the aetiology of TGCT. Exploring the functional role of the TGCT risk-associated SNPs will further elucidate the biological mechanisms involved.

Variations in testosterone pathway genes and susceptibility to testicular cancer in Norwegian men.

Imbalance between the oestrogen and androgen levels in utero is hypothesized to influence testicular cancer (TC) risk. Thus, variation in genes involved in the action of sex hormones may contribute to variability of an individual's susceptibility to TC. Mutations in testosterone pathway genes may alter the level of testosterone in vivo and hypothetically the risk of developing TC. Luteinizing hormone receptor (LHR), 5α-reductase II (SRD5A2) and androgen receptor (AR) are key elements in androgen action. A case-control study comprising 651 TC cases and 313 controls in a Norwegian population was conducted for investigation of polymorphisms in the LHR, SRD5A and AR genes and their possible association with TC. A statistical significant difference was observed in patients being heterozygous for the LHR Asn312Ser polymorphism when comparing genotypes between all TC cases and controls (OR = 0.66, 95% CI = 0.48-0.89, p(adj) = 0.049). No statistically significant difference between the histological subtypes seminoma and non-seminoma was observed. Our results may suggest a possible association between genetic variation in the LHR gene and the risk of developing TC.

CYP1A1, CYP3A5 and CYP3A7 polymorphisms and testicular cancer susceptibility.

Testicular cancer (TC) incidence is increasing worldwide, but the aetiology remains largely unknown. An unbalanced level of oestrogens and androgens in utero is hypothesized to influence TC risk. Polymorphisms in genes encoding cytochrome P450 (CYP) enzymes involved in metabolism of reproductive hormones, such as CYP1A1, CYP3A5 and CYP3A7, may contribute to variability of an individual's susceptibility to TC. The aim of this case-control study was to investigate possible associations between different CYP genotypes and TC, as well as histological type of TC. The study comprised 652 TC cases and 199 controls of Norwegian Caucasian origin. Genotyping of the CYP1A1*2A (MspI), CYP1A1*2C (I462V), CYP1A1*4 (T461N), CYP3A5*3C (A6986G) and CYP3A7*2 (T409R) polymorphisms was performed using TaqMan allelic discrimination or sequencing. The CYP1A1*2A allele was associated with 44% reduced risk of TC with each polymorphic allele [odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.40-0.78, p(trend) = 0.001], whereas the CYP1A1*2C allele was associated with 56% reduced risk of TC with each polymorphic allele (OR = 0.44, 95% CI = 0.25-0.75, p(trend) = 0.003). The decreased risk per allele was significant for seminomas (OR = 0.46, 95% CI, 0.31-0.70, p(trend) < 0.001 and OR = 0.31, 95% CI = 0.14-0.66, p(trend) = 0.002, respectively), but only borderline significant for non-seminomas (OR = 0.65, 95% CI = 0.45-0.95, p(trend) = 0.027 and OR = 0.55, 95% CI = 0.30-1.01, p(trend) = 0.052, respectively). There were no statistically significant differences in the distribution of the CYP3A5*3C and CYP3A7*2 polymorphic alleles between TC cases and controls. This study suggests that polymorphisms in the CYP1A1 gene may contribute to variability of individual susceptibility to TC.

Association between semen parameters and chance of fatherhood - a long-term follow-up study.

BACKGROUND: Evaluation of male fertility includes standard semen analysis; however, there is uncertainty about the value of sperm parameters in predicting fertility. OBJECTIVE: To evaluate the association between semen parameters and fatherhood during a long-time period. MATERIALS AND METHODS: Semen parameters (total sperm count, concentration, motility, and morphology) and sperm DNA fragmentation Index (DFI) assessed on samples collected from 195 Norwegian men from the general population in 2001/2002 were matched with information about fatherhood until 2015, obtained from the Medical Birth Register. The parameters were dichotomized as normal vs. abnormal according to the WHO reference values from 1999 and 2010. Cut-offs at 20% and 30% were used for DFI. RESULTS: Among men who had no children before 2003, those with normal progressive sperm motility had more often become fathers (WHO 1999, cut-off ≥50%, adjusted OR 2.8, 95% CI 1.3-6.1 and WHO 2010, cut-off ≥32%; aOR 4.2, 95% CI 1.1-15). Based on the WHO 1999 reference value, men with normal sperm concentration (≥20 × 106 /mL) had more often become fathers (aOR 3.1, 95% CI 1.1-8.6). Men with progressive sperm motility ≥50% and concentration ≥20 × 106 /mL did more often achieve fatherhood (aOR 8.4, 95% CI 2.1-34). For DFI, there was a borderline significance at cut-off 20% in the group of men who had ever been fathers (OR 2.7, 95% CI 1.0-7.0 p < 0.05). DISCUSSION: The results indicate that sperm progressive motility, sperm concentration, and DFI are associated with fatherhood during a longer time period, with sperm motility being most consistent. Although the sample size is relatively small and our results should be replicated in larger studies, they may be of clinical relevance. CONCLUSION: Semen parameters may have a diagnostic value not only in a short time frame but also for predicting future fertility potential.

Functions of genes related to testicular germ cell tumour development.

OBJECTIVE: Testicular germ cell tumour (TGCT) is a malignancy with a high heritable component. The inherited risk is polygenic, and around 50 susceptibility genes are identified. The functional role of the gene products for TGCT development is not well understood. The focus of this review is functional studies of genetic risk factors for TGCT derived from GCNIS and the signalling pathways involved in the pathogenesis. RECENT DEVELOPMENTS: Genome-wide association studies have identified new risk loci for TGCT and confirmed previously identified susceptibility genes. Many of these risk genes are related to male germ cell development, sex determination and genomic integrity. Gain- and loss-of-function studies in animal models and TGCT cell lines, as well as gene and protein expression studies in TGCT patient samples, have contributed to the understanding of TGCT development. KITLG-KIT signalling is of crucial importance, but several other signal transduction pathways may also play a role. Many of the risk loci are in non-coding regions, and studies have revealed that non-coding RNAs may act as oncogenes or tumour suppressors in TGCT development. CONCLUSIONS: The risk of TGCT is polygenic, and the underlying molecular mechanisms are complex. Several signalling pathways are related to TGCT development, and both proteins and non-coding RNAs may act as oncogenes or tumour suppressors. Epigenetic studies are of importance to get further knowledge about how the signalling pathways are regulated.

Mendelian randomisation analysis provides no evidence for a relationship between adult height and testicular cancer risk.

Observational studies have suggested anthropometric traits, particularly increased height are associated with an elevated risk of testicular cancer (testicular germ cell tumour). However, there is an inconsistency between study findings, suggesting the possibility of the influence of confounding factors. To examine the association between anthropometric traits and testicular germ cell tumour using an unbiased approach, we performed a Mendelian randomisation study. We used genotype data from genome wide association studies of testicular germ cell tumour totalling 5518 cases and 19,055 controls. Externally weighted polygenic risk scores were created and used to evaluate associations with testicular germ cell tumour risk per one standard deviation (s.d) increase in genetically-defined adult height, adult BMI, adult waist hip ratio adjusted for BMI (WHRadjBMI), adult hip circumference adjusted for BMI (HIPadjBMI), adult waist circumference adjusted for BMI (WCadjBMI), birth weight (BW) and childhood obesity. Mendelian randomisation analysis did not demonstrate an association between any anthropometric trait and testicular germ cell tumour risk. In particular, despite good power, there was no global evidence for association between height and testicular germ cell tumour. However, three SNPs for adult height individually showed association with testicular germ cell tumour (rs4624820: OR = 1.47, 95% CI: 1.41-1.55, p = 2.7 × 10-57 ; rs12228415: OR = 1.17, 95% CI: 1.11-1.22, p = 3.1 × 10-10 ; rs7568069: OR = 1.13, 95% CI: 1.07-1.18, p = 1.1 × 10-6 ). This Mendelian randomisation analysis, based on the largest testicular germ cell tumour genome wide association dataset to date, does not support a causal etiological association between anthropometric traits and testicular germ cell tumour aetiology. Our findings are more compatible with confounding by shared environmental factors, possibly related to prenatal growth with exposure to these risk factors occurring in utero.

Fatty acid composition of spermatozoa is associated with BMI and with semen quality.

High body mass index (BMI) is negatively associated with semen quality. In addition, the composition of fatty acids of spermatozoa has been shown to be important for their function. The aim of the study was to examine the association between BMI and the composition of spermatozoa fatty acids in men spanning a broad BMI range. We also analysed the relation between fatty acid composition of spermatozoa and semen characteristics, and the relationship between serum fatty acids and spermatozoa fatty acids. One hundred forty-four men with unknown fertility status were recruited from the general population, from couples with identified female infertility and from morbid obesity centres. Standard semen analysis (WHO) and sperm DNA integrity (DFI) analysis were performed. Fatty acid compositions were assessed by gas chromatography. When adjusted for possible confounders, BMI was negatively associated with levels of sperm docosahexaenoic acid (DHA) (p < 0.001) and palmitic acid (p < 0.001). The amount of sperm DHA correlated positively with total sperm count (r = 0.482), sperm concentration (r = 0.469), sperm vitality (r = 0.354), progressive sperm motility (r = 0.431) and normal sperm morphology (r = 0.265). A negative association was seen between DHA levels and DNA fragmentation index (r = -0.247). Levels of spermatozoa palmitic acid correlated positively with total sperm count (r = 0.227), while levels of linoleic acid correlated negatively (r = -0.254). When adjusted for possible confounders, only the levels of arachidonic acid showed positive correlation between spermatozoa and serum phospholipids (r = 0.262). Changes in the fatty acid composition of spermatozoa could be one of the mechanisms underlying the negative association between BMI and semen quality. The relationship between fatty acids of spermatozoa and serum phospholipids was minor, which indicates that BMI affects fatty acid composition of spermatozoa through regulation of fatty acid metabolism in the testis. The role of dietary intake of fatty acids on the spermatozoa fatty acid composition remains to be elucidated.

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats.

The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [(14)C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-(14)C]20:5n-3 to [3-(14)C]22:6n-3 was more efficient than that of [1-(14)C]20:4n-6 to [3-(14)C]22:5n-6. At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM). The conversion of [1-(14)C]22:5n-3 to [1-(14)C]22:6n-3 was 1.7 times more efficient than that of [1-(14)C]22:4n-6 to [1-(14)C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-(14)C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-(14)C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-(14)C]20:4n-6 or [1-(14)C]22:4n-6 to [(14)C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-(14)C]20:5n-3 and [1-(14)C]22:5n-3 to [(14)C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.

A comparative study of the metabolism of n-9, n-6 and n-3 fatty acids in testicular cells from immature rat.

Dietary 18 and 20-carbon fatty acids of the n-6 and the n-3 families are metabolized to 22:5,n-6 and 22:6,n-3 by a sequence of specific desaturases and chain elongation via 24-carbon intermediates. This pathway is regulated so that more 22:6,n-3 than 22:5,n-6 is found in the tissues. Rat testis is an exception since 22:5,n-6 is present in large proportions in this organ. Therefore rat testis appears to be interesting for studies of the detailed synthesis of 22:5,n-6 compared with that of 22:6,n-3. By using fresh preparations of rat testicular cells from 19-day-old rats enriched in Sertoli cells, we compared the metabolism of 1-14C-labelled n-3, n-6 and n-9 fatty acids. The testicular cells actively synthesized 22:6,n-3 and 22:5, n-6, but not 22:4,n-9 from the 18 and 20-carbon precursors. Of 200 mol 14C-labelled C18 and C20 fatty acids added initially, approximately 20-40 mol were found as 24-carbon intermediates after 24 h of incubation. This indicates that the balanced capacity of elongation, desaturation and chain shortening favours the accumulation of 24-carbon intermediates in these cells. One exception was [1-14C]20:3,n-9 which was efficiently elongated to 22:3,n-9 but not to C24 fatty acids. Our data suggests that the poor elongation of n-9 fatty acids from C22- to C24 may be an important hindrance in the synthesis of 22:4,n-9. The efficient synthesis of 22:5,n-6 may also partly explain why this is the major 22-carbon fatty acid in rat testis.

The alpha-subunit mRNAs for Gs and Go2 are differentially regulated by protein kinase A and protein kinase C in rat Sertoli cells.

In the present study, we have examined regulatory effects of protein kinase A and protein kinase C activation by 8-CPTcAMP and TPA, respectively, on mRNAs for various G protein alpha-subunits and corresponding immunoreactive proteins in rat Sertoli cells. Gs alpha and Go alpha mRNA levels were transiently increased 1.5-fold and 4-fold, respectively, by 8-CPTcAMP in cultured Sertoli cells. This up-regulation of mRNAs for Gs alpha and Go alpha was also observed when Sertoli cells were incubated in the presence of FSH. When protein synthesis was inhibited by cycloheximide, the cAMP-mediated stimulation of Gs alpha mRNA was abolished, whereas Go alpha mRNA was superinduced to a 50- to 100-fold higher level than basal. Activation of protein kinase C with TPA had a strong, synergistic effect on cAMP-mediated stimulation of Gs alpha mRNA, whereas the cAMP-mediated stimulation of Go alpha mRNA was completely blocked. Surprisingly, changes in mRNA levels were not accompanied by any alterations in the levels of immunoreactive Gs alpha and Go alpha proteins.

Elevated expression of lanosterol 14alpha-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats.

Mammalian CYP51 encodes lanosterol 14alpha-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14alpha-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII-XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observed. The rat male germ cell-specific CYP51 transcript is translated in vitro to two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher in protein extracts of testes and germ cells of sexually mature rats than in prepubertal animals, in which postmeiotic germ cells are not yet present. This shows increased capacity for the production of MAS sterols by male germ cells that have already completed meiosis, suggesting that they serve a role different from meiosis activation.

Serum factors induce messenger ribonucleic acid levels for cellular retinol-binding protein in rat Sertoli cells.

This report shows that serum factors dramatically increase the levels of mRNA for cellular retinol-binding protein (CRBP) in cultured rat Sertoli cells. Incubation of rat Sertoli cells (0-24 h) with 10% fetal calf serum (FCS) was associated with a time-dependent increase in CRBP mRNA levels. A significant increase (6-fold) was observed after 3 h of incubation. Maximal levels (15- to 50-fold) were reached after 9-12 h and were maintained for as long as serum was present. The effect was concentration dependent, with maximal induction at 10% FCS. Removal of FCS resulted in a decline in the CRBP mRNA levels, with a t1/2 of approximately 7 h. The CRBP mRNA stimulating activity (CMSA) was completely removed from FCS by precipitation with 5% trichloroacetic acid, but was only partly (50%) inhibited by heating at 100 C or trypsin treatment. Removal of retinol from FCS by repeated ether extractions did not alter the CMSA of FCS. Both the induction and degradation of CRBP mRNA were inhibited by the protein synthesis inhibitor cycloheximide. A nuclear protein binding to the 5'-flanking region of the CRBP gene was detected in nuclear extracts from untreated Sertoli cells, but not in nuclear extracts from Sertoli cells treated with 10% FCS for 3 h. Thus, serum factors, different from retinoids, dramatically stimulate the levels of CRBP mRNA in rat Sertoli cells. This is associated with the loss of protein binding to the 5'-flanking region of the CRBP gene.

Cell-specific expression of Gq/11 protein and mRNA in rat seminiferous tubules.

As part of a study to elucidate the involvement of G proteins in signal transduction in testicular cells, we have examined the cellular localization of Gq/11 within the seminiferous tubules. The somatic cells (Sertoli cells, peritubular cells) contain high amounts of both Gq/11 alpha mRNA and immunoreactive protein. In contrast, very low levels of these G proteins and the corresponding mRNAs are present in the germ cells (pachytene spermatocytes, round spermatids). Thus, in the germ cells, receptor-regulated inositol phospholipid hydrolysis is not likely to be regulated via Gq/11, but rather through the Go protein, which has been previously shown to be abundant in rat germ cells. Since the somatic cells are nearly devoid of Go, the Gpp(NH)p-stimulated phospholipase C in these cells is probably regulated by Gq and/or G11.

Residual bodies and IL-1alpha stimulate expression of mRNA for IL-1alpha and IL-1 receptor type I in cultured rat Sertoli cells.

The cytokine interleukin (IL)-1alpha may be produced both by Sertoli cells and immature male germ cells from rat and is thought to play a role in autocrine and/or paracrine regulation of the spermatogenesis. The localization of IL-1 receptors in seminiferous tubules is unknown. In this study we found a constitutive expression of IL-1 receptor type I (IL-I RI) mRNA in cultured Sertoli cells and peritubular cells from rat, whereas no such transcripts were observed in immature germ cells (pachytene spermatocytes and round spermatids). An autostimulation of IL-1alpha mRNA synthesis has previously been described in other cell types. Stimulation of Sertoli cells with recombinant IL-1alpha for 0-7 h resulted in a rapid increase in both IL-1alpha and IL-1 RI mRNA. When Sertoli cells were cultured with residual bodies for 0-48 h, mRNA levels for both IL-1alpha and IL-1 RI were increased in a biphasic manner. We suggest that phagocytosis of residual bodies triggers an autocrine IL-1alpha loop in Sertoli cells where both IL-1alpha and one of its receptors are stimulated.