One-year results of a multicenter controlled clinical trial of triamcinolone in pars plana vitrectomy

Purpose To evaluate the 1-year results of using triamcinolone acetonide (TA) in pars plana vitrectomy (PPV).
Introduction
Recently, vitrectomy has been used to treat various vitreoretinal diseases. It still remains a demanding procedure requiring a skilled and experienced surgeon, despite advances in surgical instrumentation and techniques. One major difficulty with this procedure is the transparency of the vitreous. Intra-operative triamcinolone acetonide (TA) has been used to address this problem, as it can be injected into the vitreous to visualize the posterior hyaloid or the internal limiting membrane (ILM) [1, 2]. Several previous reports have demonstrated the usefulness of TA as an adjunct to pars plana vitrectomy (PPV) [3–13]. Although their findings supported the use of TA-assisted PPV, these studies involved relatively small numbers of eyes and were not randomized. In addition, more recent reports on the use of intravitreous TA in patients not undergoing vitrectomy raised concerns about the risks of elevated intra-ocular pressure (IOP) [14–19].
We therefore undertook a multicenter prospective controlled study of TA-assisted pars plana vitrectomy (PPV). Considering the enduring nature of the effect of PPV on ocular tissue, it is important to investigate the long-term results in order to evaluate TA-assisted PPV. Moreover, the crucial issue of the effects on patients’ vision must be explored before TA-assisted PPV can be recommended. The current report presents the 1-year results of the use of TA in PPV, and evaluates the procedure in light of the aforementioned issues.
Methods
Study design, randomization and sample size
The prospective controlled clinical trial took place at eight hospitals in Japan as previously described (see Acknowledgements below) [13].
The patients were assigned to the two groups using a single-blind quasi-randomization approach within the participating clinical centers. Our preliminary study showed that the most important factor influencing the surgical outcome was not the nature of the disease for which PPV was used, but rather the hospital in which the surgery was performed (data not shown). To minimize this bias, equal numbers of patients from each hospital were assigned to each of the study groups. Thus, the patients were assigned by a single-blind quasi-randomization within the participating clinical centers. Briefly, after the patients passed the inclusion/exclusion criteria and gave consent for the study, they were numbered serially at each hospital, and alternate numbers were assigned to the TA-assisted and conventional PPV groups, respectively, as previously described [13]. Data analysis was performed by an intent-to-treat analysis method. To conceal the allocations at the participating hospitals, the surgeons were not informed of the group to which a patient had been assigned until immediately before surgery.
This is the second report of controlled clinical trial of intraocular TA in PPV. Primary outcome was whether the use of TA might reduce the incidence of intraoperative complications. The sample size was determined based upon our preliminary study, and the method has been described elsewhere [13]. Briefly, the incidence of intraoperative retinal breaks was found to be approximately 15% in the participating hospitals. The sample size calculation was based on our pilot study data, and was aimed to detect a difference in short-term postoperative complications as reported previously [13]
Eligibility criteria and surgery
Institutional review board (IRB)/ethics committee approval was obtained from all of the participating clinical establishments, such that all patients undergoing PPV at these hospitals were eligible to enroll in the study. Each patient was fully informed about the nature of the treatment, and provided written consent. The following groups were excluded: (1) patients with diagnoses that included macular holes; patients with uveitis, (2) patients with phakic lenses, (3) patients who were known to be steroid responders, to have glaucoma, or to have apparent infectious endophthalmitis, and (4) patients who were undergoing a second PPV. For ethical reasons, if at the time of surgery a surgeon felt that TA was necessary to achieve a successful PPV outcome, it was included in the procedure; for the purposes of the intent-to-treat analysis, these cases were included within the conventional PPV group.
The details of the surgical protocols varied between the hospitals, but the basic procedure was similar. PPV was performed according to a previously reported methodology, either with or without the use of a TA suspension prepared as described elsewhere [2, 13]. The recording of the operations, control of surgical quality, use of antibiotics, and termination of the study have been described elsewhere [13].
During the follow-up period, additional surgeries were performed at each hospital as and when they were needed. For example, filtering surgeries were performed when the intra-ocular pressure (IOP) remained at an uncontrollably high level (25 mmHg or greater) even with full medication. Full medication included the use of all latanoprost, beta-blocker, and dorzolamide hydrochloride eyedrops and acetazolamide medicine.
Data collection and documentation were performed as previously described [13]. For the purposes of this study, the adverse events related to surgery were defined as follows: retinal breaks, macular pucker, retinal fibrous membrane formation, retinal detachment, vitreous hemorrhage (VH), rubeosis iridis, optic-disc damage, corneal diseases, and after cataract. The serious adverse events related to surgery were defined as follows: infectious endophthalmitis, retinal degeneration, unexplained optic-disc damage, and unexplained deterioration of vision.
Masking and outcome assessment
The study records were collected by the principal investigator (TS) up until November 2006. After checking the records for harmful events, the data were sent to the controllers (YN) at Kyushu University. The remaining investigators were not permitted access to any information on the outcomes before completion of the analysis. Visual acuity was measured by ophthalmic technicians using the Snellen high-contrast acuity test. IOP was also measured by the ophthalmic technicians, and the findings were reviewed by the physicians. IOP was measured with pneumotonometer. The ophthalmic technicians were not informed of the purpose of the study or the assignment schedule. Anti-glaucoma eye drops and/or acetazolamide were used when an IOP of 22 mmHg or higher persisted for 3 days.
Statistical analysis
The visual acuity was converted to the logarithm of the minimum angle of resolution (LogMAR), and the baseline value was compared with those at 1 week, 1 month, 3 months, 6 months, and 1 year. The eyes were categorized into the following three groups: (1) the improved group, in which patients showed a 0.3 logMAR or greater improvement of visual acuity at the final examination compared with baseline, (2) the deteriorated group, in which patients showed a 0.3 logMAR or worse deterioration of visual acuity at the final examination compared with baseline, and (3) the unchanged group, in which patients did not meet the criteria of either the improved or the deteriorated group.
A Kaplan-Meier survival analysis was used to estimate the probability of improved visual acuity with time after surgery. The improvement or deterioration was analyzed by the log rank test. The earliest time point after surgery at which the events in each category were reported was designated as the time point of each event.
We also estimated the multiple-adjusted odds ratio (OR) and the 95% confidence interval (95%CI) both for TA use and for the other variables, using a logistic regression model. The parameters that were adjusted for included age, gender, and disease diagnosis, which was categorized into the following five groups: proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), and others. A diagnosis of PDR was used as a reference. Even when a single eye received multiple additional surgeries, it was counted as one eye. All statistical analyses were performed using SAS® (Proprietary Software Release 8.2; SAS Institute Inc., Cary, NC, USA). The differences in diagnosis or surgical intervention between the two groups were compared using a two-tailed Chi-squared test with the Yates’ correction. P values < 0.05 were considered statistically significant.
Results
Number of eyes
In total, 920 patients were initially registered for PPV during this period. Among these, 146 eyes were not included in the study because they did not meet the eligibility criteria or because the patient declined to participate. In total, 774 eyes were randomized, with 391 eyes (from 216 male and 175 female patients) assigned to the TA-assisted PPV group and 383 eyes (201 male and 182 female patients) assigned to the conventional PPV group. The mean ages of the patients were 63.5 ± 12.1 years in the TA-assisted PPV group and 63.0 ± 11.3 years in the conventional PPV group. In total, 19 eyes that were originally assigned to the conventional PPV group received unplanned TA based on surgical judgment. The 1-year post-operative follow-up was completed for a total of 305 eyes in the TA-assisted PPV group (78.0%) and 295 eyes in the conventional PPV group (77.0%). The overall follow-up rate was 77.5% (Fig. 1).
Fig. 1Schematic diagram of the study. TA: triamcinolone acetonide, PPV: pars plana vitrectomy
Diagnoses
The diagnoses for each group are shown in Table 1. In both groups, PDR accounted for the largest number of cases, followed by RRD. In both groups, the pattern at the 1-year follow-up was similar to that at the baseline, and the different diagnoses had significantly different distributions within the two groups: the TA-assisted PPV group had a relatively larger number of patients with DME (P = 0.018 at baseline, P = 0.006 at 1-year follow-up) and a smaller number of patients with epiretinal membrane (ERM) formation (P = 0.031 at baseline, P = 0.008 at 1-year follow-up).
Table 1Diseases of each group at baseline and 1 year after surgery TA-assisted PPVConventional PPVBaseline1 yearbaseline1 yearN (%)N (%)N (%)N (%)AMD7 (1.8%)3 (1.0%)3 (0.8%)3 (1.0%)BRVO50 (12.8%)41 (13.4%)37 (9.7%)29 (9.8%)CRVO11 (2.8%)9 (3.0%)7 (1.8%)6 (2.0%)DME67 (17.1%)60 (19.7%)42 (11.0%)34 (11.5%)ERM24 (6.1%)19 (6.2%)41 (10.7%)37 (12.5%)Lens luxation16 (4.1%)11 (3.6%)16 (4.2%)12 (4.1%)Macroaneurysm10 (2.6%)8 (2.6%)9 (2.3%)4 (1.4%)PDR113 (28.9%)85 (27.9%)131 (34.2%)101 (34.2%)RRD80 (20.5%)59 (19.3%)77 (20.1%)54 (18.3%)VH6 (1.5%)6 (2.0%)14 (3.7%)11 (3.7%)Others7 (1.8%)4 (1.3%)6 (1.6%)4 (1.4%)total383 (100%)305 (100%)391 (100%)295 (100%)TA; triamcinolone acetonide: PPV; pars plana vitrectomy: AMD; age-related macular degeneration: BRVO;branch retinal vein occlusion: CRVO: central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment: VH; vitreous hemorrhage.
There were no significant differences between the two groups in the usage of surgical methods, such as cataract surgery (phacoemulsification P = 0.724 and pars plana lensectomy P = 0.272), ILM peeling (P = 0.075) and endolaser-photocoagulation (P = 0.413), or in the use of post-operative tamponade (sulfur hexafluoride gas P = 0.810, octa-fluorepropane gas P = 0.756, room air P > 0.999, and silicone oil P = 0.831) [13].
Visual acuity
The visual acuity in both groups improved over time. In the TA-assisted PPV group, 322 eyes were categorized as improved, 21 were categorized as unchanged, and 48 were categorized as deteriorated. In the conventional PPV group, 312 eyes were categorized as improved, 45 eyes were categorized as unchanged, and 26 eyes were categorized as deteriorated. The log-rank test revealed no significant difference between the two groups regarding the improvement (P = 0.98) or deterioration (P = 0.26) of visual acuity. The logistic regression model also showed that the intra-operative use of TA was not a significant factor for the improvement of visual acuity (P = 0.91) after adjusting for age, gender, and diagnosis (Table 2). Neither gender (P = 0.54) nor age (P = 0.31) was a significant factor for the improvement of visual acuity. However, PDR diagnosis was a significant factor for the improvement of visual acuity compared with DME (OR = 0.63, 95%CI = 0.478–0.817, P = 0.0006; Table 2).
Table 2Factors affecting improvement of visual acuityVariable (no. of improved eyes/total eyes)Odds ratio (95%CI)P-valueTA-assisted PPV (322/391) vs conventional PPV (312/383)1.10 (0.860–1.183)0.91Age1.00 (0.991–1.005)0.54Gender (male vs female)0.92 (0.785–1.080)0.31DiseasesRD vs PDR1.04 (0.834–1.340)0.71DME vs PDR0.63 (0.478–0.817)0.0006*RVO vs PDR1.21 (0.938–1.561)0.14ERM vs PDR0.78 (0.568–1.061)0.11Others vs PDR1.21 (0.924–1.579)0.17CI; confidence interval: TA; triamcinolone acetonide: PPV; pars plana vitrectomy: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment. * statistically significant
Post-operative complications
Subgroup analysis
In terms of post-operative complications, there was not any statistically significant difference. Fibrous ERM was observed in five eyes (1.4%) in the conventional PPV group, but in only one eye (0.3%) in the TA-assisted PPV group. A subgroup analysis showed that this difference was not significant (P = 0.10). Among the other complications, an IOP increase was found more frequently in the TA-assisted PPV group (23 eyes, 5.9%) than in the conventional PPV group (13 eyes, 3.4%), although this difference was not statistically significant (P = 0.10). There were no significant differences in the other complications between the two groups (Table 3).
Table 3Post-operative complications in each group TA-assisted PPV (total 391 eyes): n (%)Conventional PPV (total 383 eyes): n (%)P-value*After cataract18 (4.6%)17 (4.4%)0.91Macular pucker23 (5.9%)22 (5.7%)0.93IOP increase23 (5.9%)13 (3.4%)0.10Retinal detachment9 (2.3%)9 (2.3%)0.96Vitreous hemorrhage22 (5.6%)29 (7.8%)0.28Fibrous membrane1 (0.3%)5 (1.4%)0.10Iris synechiae6 (1.5%)4 (1.0%)0.55Others8 (2.0%)12 (3.1%)0.34TA; triamcinolone acetonide: PPV; pars plana vitrectomy: PPL; pas plana lensectomy: IOP; intraocular pressure: * Chi-square test with the Yates’ correction.
Additional surgery
Although the filtering surgery was more performed in TA-assisted PPV and the removal of vitreous hemorrhage was more performed in conventional PPV, there was not any statistically significant difference between the two groups with respect to causes of additional surgery after PPV. In the TA-assisted PPV group, 22 surgeries were performed (excluding filtering surgery). The most common reasons for additional surgery were removal of silicone oil (11 eyes), followed by retinal detachment (eight eyes), VH (six eyes), and others (seven eyes). In the conventional PPV group, 26 surgeries were performed (excluding filtering surgery). The most common reasons were VH (12 eyes), followed by retinal detachment (nine eyes), removal of silicone oil (seven eyes), and others (six eyes). Although there was a tendency for VH to be less common in TA-assisted PPV eyes, no significant difference was found in comparison to conventional PPV (P = 0.13; Table 4). Filtering surgery was more frequently performed in the TA-assisted PPV group (15 cases, 3.8%) than the conventional PPV group (seven cases, 1.8%), although this difference was not significant (P = 0.10). Ten eyes received multiple additional surgeries in TA-assisted PPV group and seven eyes in conventional PPV group.
Table 4Causes of additional surgery after PPV TA-assisted PPV (total 391 eyes): n (%)Conventional PPV (total 383 eyes) : n (%)P-value*Silicone removal11 (2.8%)7 (1.8%)0.36Retinal detachment8 (2.0%)9 (2.3%)0.77Vitreous hemorrhage6 (1.5%)12 (3.1%)0.14Filtering surgery15 (3.8%)7 (1.8%)0.10Others7 (1.8%)6 (1.6%)0.81Total37 (9.5%)✝32 (8.6%)✝✝0.68TA; triamcinolone acetonide: PPV; pars plana vitrectomy: * Chi-square test with the Yates’ correction: ✝ Ten eyes received multiple additional surgeries: ✝✝ Seven eyes received multiple additional surgeries.
The survival curve produced by the log-rank test showed that the incidence of additional surgery was not significantly different between the two groups (P = 0.45). The logistic regression test also showed that the use of TA was not a significant factor for additional surgery after adjustments for gender, age, and diagnosis. However, a diagnosis of PDR was a significant factor for additional surgery in comparison to DME (P = 0.005), RVO (P = 0.006), and ERM (P = 0.02; Table 5). None of the variables were related to the requirement for filtering surgery (Table 6).
Table 5Factors affecting additional surgeryVariable (no. of additional surgery/total)Odds ratio (95%CI)P-valueTA-assisted PPV (37/391) vs conventional PPV (32/383)1.23 (0.797–1.911)0.35Age0.97 (0.950–0.984)0.0001*Gender (male vs female)1.15 (0.733–1.797)0.55DiseasesRD vs PDR0.60 (0.345–1.029)0.06DME vs PDR0.29 (0.122–0.681)0.005*RVO vs PDR0.23 (0.084–0.657)0.006*ERM vs PDR0.087 (0.012–0.631)0.02*Others vs PDR0.64 (0.299–1.374)0.25CI; confidence interval: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment. * statistically significantTable 6Factors affecting filtering surgeryVariable (no. of filtering surgery/total)Odds ratio (95%CI)P-valueTA-assisted PPV (12/391) vs conventional PPV (5/383)1.74 (0.708–4.265)0.23Age0.98 (0.941–1.018)0.28Gender (male vs female)1.81 (0.696–4.728)0.22DiseasesRRD vs PDR–0.99DME vs PDR–0.99RVO vs PDR–0.99ERM vs PDR–0.99Others vs PDR0.83 (0.266–2.573)0.74CI; confidence interval: TA; triamcinlone acetonide: PPV; pars plana vitrectomy: RVO; branch retinal vein occlusion + central retinal vein occlusion: DME; diabetic macular edema: ERM; epiretinal membrane: PDR; proliferative diabetic retinopathy: RRD; rhegmatogenous retinal detachment
Serious adverse events
None of the serious adverse events related to surgery defined in this study (such as retinal degeneration, endophthalmitis, an unexplained decrease in visual acuity, or optic-disc atrophy) were observed in either group during the 1-year follow-up.
Discussion
The first report on this study showed that the intra-operative use of TA significantly decreased the incidence of intra-operative complications associated with PPV, and no adverse events related to surgery were found over a 3-month observation period [13]. A decreased incidence of intra-operative complications is beneficial; however, the critical issue is whether the surgery improves the patients’ vision. We therefore followed up the visual acuity, which is a crucial factor influencing vision, for 1 year after surgery. Our results revealed no significant difference in visual acuity between the TA-assisted PPV group and the conventional PPV group after 1 year. As described in the earlier report, which analyzed data from the same eyes, intra-operative retinal breaks and retinal detachment were significantly less frequent in the TA-assisted PPV group than in the conventional PPV group. Nonetheless, the decreased incidence of intra-operative complications did not significantly affect the visual acuity after 1 year. This finding can be explained by the following facts. First, surgical skill is the most important factor for surgical success. As all of the operations in this study were performed by qualified and experienced surgeons, the intra-operative complications were managed so as not to cause serious post-operative complications. Thus, the influence of intra-operative complications on visual acuity might have been minimal. Second, a patient’s vision after vitrectomy might be more strongly affected by a disease diagnosis than by intra-operative complications. Indeed, eyes with PDR were significantly more likely to show improved visual acuity compared with baseline than eyes with DME in the current study. As our present case series included various diseases, the impact of intra-operative complications on post-operative visual acuity might have been masked by another strong factor, such as disease diagnosis, even after adjustments. Third, the eyes in the current study were primarily chosen for the detection of intra-operative retinal breaks, based on the hypothesis that the intra-operative use of TA might decrease their incidence from 15% to 8% [13]. Thus, the sample size might not have been large enough to detect the superiority or inferiority of TA-assisted PPV. For example, in order to detect the difference of post-operative improvement of vision in TA-assisted PPV and conventional PPV based upon the present results, approximately 29,315 cases in each group are necessary with α power of 0.05 and 1-β of 0.8. However, considering the relative stability of the visual acuity observed here, we are confident that the intra-operative use of TA does not have a positive or negative effect on postoperative visual acuity for 1 year.
Additional surgeries carry associated risks and can be costly; they are thus important when justifying the intra-operative use of TA in PPV. The incidence of additional surgery after vitrectomy was found to be equivalent using both methods. Considering the IOP increase after surgery, the opportunity to use anti-glaucoma eye drops was high in the TA-assisted PPV group [13], and the number of filtering surgeries performed during 1 year after TA-assisted PPV was also high; however, the incidence of additional surgeries was not statistically different between the groups. Several previous reports showed that intravitreous TA injection required filtering surgery, and its frequency was less than 1% of all treated eyes [18, 19]. In contrast to intravitreous TA injection, most of the TA was removed from the eye at the end of the surgery. Thus, it was plausible that the necessity of filtering surgery in TA-assisted PPV was not significant. The other causes of additional surgery occurred at similar frequencies, and there were no significant differences between the groups. A diagnosis of PDR was a significant factor for the requirement for additional surgery, as it was for the improvement of visual acuity. Furthermore, in our earlier report on the 3-month follow-up study, a diagnosis of PDR was found to be a significant risk factor for the occurrence of intra-operative complications. PDR eyes thus appear to have several poor prognostic factors, consistent with the present results.
With regard to the post-operative complications, pre-retinal fibrosis appeared to be less common in the TA-assisted PPV group than in the conventional PPV group, although this was not statistically significant. This advantage of TA-assisted PPV has been described in previous reports [3, 5–9]. The intra-operative use of TA visualizes the posterior hyaloid clearly, which facilitates its removal [6, 7]; as the residual posterior hyaloid can act as a scaffold for pre-retinal fibrous-membrane formation, its removal could reduce the incidence of post-operative pre-retinal fibrosis [5–7]. In the current study group, the removal of the residual posterior hyaloid was carried out according to the surgeons’ preference—some surgeons removed it completely, while others did not. This might have obscured any potential advantage of the intra-operative use of TA in inhibiting post-operative pre-retinal fibrosis.
There were clear limitations to our analysis. The number of cases in each study group was primarily chosen to allow the detection of intra-operative complications; thus, the significance of the intra-operative use of TA might only become clear when studying larger numbers of patients. In addition, the sample size was too small to detect the risk of endophthalmitis. The incidence of endophthalmitis related to PPV was 0.046% in the conventional PPV group and 0.053% in the TA-assisted PPV group [20, 21]. An analysis of a greater number of cases will thus be necessary to evaluate the risk of endophthalmitis related to TA-assisted PPV. However, the present sample size was large enough to detect several factors that affected post-operative visual acuity or the need for additional surgery. Another limitation was that the post-operative visual acuity was strongly influenced by the diagnosis of disease. Eyes with simple VH clearly had a greater chance of surgery improving vision above the baseline level than did eyes with complicated PDR. In order to evaluate the influence of the intra-operative use of TA on visual acuity more precisely, we are currently performing a study on eyes with the same disease diagnosis. Furthermore, the subgroup of DME or ERM in each group was not completely matched. The interpretation of the results should be done carefully. Additionally, caution must be applied when making generalizations on the basis of our current results, and their application to clinical practice must be undertaken with care. Other factors, such as invasiveness and cost, are also important in seeking justification for this treatment. The success of surgery depends upon various factors, and surgical skill is critical. When considering the likelihood of retinal breaks and their recovery without damaging vision, comparisons of the hazard ratios of TA-assisted PPV and conventional PPV would be valid only if made by individuals who qualify as PPV specialists.
In summary, the intra-operative use of TA in PPV did not affect visual acuity over 1 year. As negative or adverse events were not observed in this case series, a more detailed study is warranted to establish the value of TA in PPV.

steroid
retinal detachment
diabetic retinopathy
proliferative vitreoretinopathy
adjuvant


The evolution of tooth wear indices

Tooth wear—attrition, erosion and abrasion—is perceived internationally as an ever-increasing problem. Clinical and epidemiological studies, however, are difficult to interpret and compare due to differences in terminology and the large number of indices that have been developed for diagnosing, grading and monitoring dental hard tissue loss. These indices have been designed to identify increasing severity and are usually numerical. Some record lesions on an aetiological basis (e.g. erosion indices), others record lesions irrespective of aetiology (tooth wear indices); none have universal acceptance, complicating the evaluation of the true increase in prevalence reported. This article considers the ideal requirements for an erosion index. It reviews the literature to consider how current indices have evolved and discusses if these indices meet the clinical and research needs of the dental profession.
Introduction
There is both a clinical and a scientific need to be able to measure tooth wear, and the literature abounds with many methods which can be broadly divided into quantitative and qualitative in nature. Quantitative methods tend to rely on objective physical measurements, such as depth of groove, area of facet or height of crown. Qualitative methods, which rely on clinical descriptions, can be more subjective if appropriate training and calibration are not carried out but which, with correct safeguards, can be valuable epidemiological tools. In a clinical intra-oral examination, there will be an inclination towards descriptive assessment measures, such as mild, moderate or severe, rather than quantitative measurement, which is easier to perform reliably on a model or in the laboratory. Such methods tend to be more sensitive but do not lend themselves readily to clinical use—especially in epidemiology, where fieldwork data collection is often carried out in an environment lacking sophisticated equipment.
Quantitative and qualitative methods typically utilise grading or scoring systems designed to identify increasing severity or progression of a condition; these are described as indices and are usually numerical. An ideal index should be simple to understand and use, clear in its scoring criteria and be demonstrably reproducible. Its application should be useful for research into the aetiology, prevention and monitoring of a condition, essentially being an epidemiological and clinical tool.
Review of the literature reveals the fact that many different tooth wear indices have been developed for clinical and laboratory use all over the world. Unfortunately, the production of so many indices does not allow for ready comparison of results between different working groups, and this is especially important in epidemiology when trying to define the prevalence of a condition. Confusion is further generated in the literature as the majority of researchers, in their attempts to quantify the amount of tooth tissue loss due to tooth wear, have historically concentrated on one aetiology only, and these indices tend to be surface limited. Often, the wear patterns described do not appear to reflect the aetiology suggested, and this relates to lack of uniformity with tooth wear terminology and translation errors. Many diagnostic indices do not properly reflect the morphological defects, and there is little international standardisation. All of these factors complicate the comparison of data and evaluation of the efficacy of preventive and therapeutic measures.
The literature identifies separate indices for use in clinical and laboratory situations and specific indices for attrition, abrasion, erosion and multifactorial tooth wear. There are common threads to all of the indices, such as descriptive diagnostic criteria and criteria for quantifying the amount of hard tissue loss. These generally consider the size of the affected area—as a proportion of a sound surface and/or the depth of tissue loss—often expressed as a degree of dentine exposure.
The clinical measurement of erosion
The earliest indices shared common, arbitrary criteria, relying on descriptive terms such as slight, mild, moderate, severe and extensive. Restarski et al. [26] developed a six-point grading system to evaluate the severity of erosive destruction observed on the lingual surfaces of rat and puppy molars, but concerns were raised with regards to reproducibility. With vague criteria definitions, variability in recording is expected. Each animal was allocated a total score, calculated by summing the mean molar quadrant scores. Whilst producing simple data for analysis, it is acknowledged that averaging scores in this manner leads to the loss of much data. If the number of teeth severely affected is small, the erosion score will be low; but this could mask a significant, localised clinical problem [27].
Eccles [9] originally classified lesions broadly as early, small and advanced, with no strict criteria definitions, thus allowing wide interpretation. Later, the index was refined and expanded, with greater emphasis on the descriptive criteria [10]. It was presented as a comprehensive qualitative index, grading both severity and site of erosion due to non-industrial causes, and is considered as one of the cardinal indices from which others have evolved. In essence, it breaks down into three classes of erosion, denoting the type of lesion, assigned to four surfaces, representing the surface where erosion was detected (Table 1).
Table 1Eccles index for dental erosion of non-industrial origin [10]ClassSurfaceCriteriaClass I Early stages of erosion, absence of developmental ridges, smooth, glazed surface occurring mainly on labial surfaces of maxillary incisors and caninesClass IIFacialDentine involved for less than one third surface; two typesType 1 (commonest): ovoid–crescentic in outline, concave in cross section at cervical region of surface. Must differentiate from wedge shaped abrasion lesionsType 2: irregular lesion entirely within crown. Punched out appearance, where enamel is absent from floorClass IIIaFacialMore extensive destruction of dentine, affecting anterior teeth particularly. Majority of lesions affect a large part of the surface, but some are localised and hollowed outClass IIIbLingual or palatalDentine eroded for more than one third of the surface area. Gingival and proximal enamel margins have white, etched appearance. Incisal edges translucent due to loss of dentine. Dentine is smooth and anteriorly is flat or hollowed out, often extending into secondary dentineClass IIIcIncisal or occlusalSurfaces involved into dentine, appearing flattened or with cupping. Incisal edges appear translucent due to undermined enamel; restorations are raised above surrounding tooth surfaceClass IIIdAllSeverely affected teeth, where both labial and lingual surfaces are extensively involved. Proximal surfaces may be affected; teeth are shortened
Greater accuracy was introduced by Xhonga and Valdmanis [33] who divided erosions into four levels by measurement with a periodontal probe: none, minor (less than 2 mm), moderate (up to 3 mm) and severe (greater than 3 mm). They further differentiated types of erosion by morphological descriptions, such as wedge, saucer, groove and atypical. They did not address the problem of inter- or intra-examiner variability.
The clinical measurement of tooth wear
It is perhaps significant that the earliest index documented by Broca [4] and used as a foundation for the development of further indices graded horizontal or oblique patterns of occlusal wear without presupposing the aetiology. Smith and Knight [30] introduced the more general concept of measuring tooth wear per se, irrespective of the cause, and since then more recent indices have been developed or modified from Smith and Knight that do not rely on a prior diagnosis and are more clinically relevant. Most of these stress the importance of user training sessions and calibration exercises.
Smith and Knight [30] took Eccles’ ideas a stage further, producing the tooth wear index (TWI), a comprehensive system whereby all four visible surfaces (buccal, cervical, lingual and occlusal–incisal) of all teeth present are scored for wear, irrespective of how it occurred (Table 2). This avoids the confusion associated with terminology and translation or differences in opinion for diagnosis of aetiology based on clinical findings. Guidelines for using the criteria were produced in a booklet by the authors to aid training and standardisation with other investigators; in cases of doubt, the lowest score is given. Complete enamel loss (score 4) may, however, be misleading, as there is almost always a rim of enamel at the worn surface margins—the colloquial “enamel halo.”
Table 2Smith and Knight tooth wear index [30]ScoreSurfaceCriteria0B/L/O/INo loss of enamel surface characteristicsCNo loss of contour1B/L/O/ILoss of enamel surface characteristicsCMinimal loss of contour2B/L/OLoss of enamel exposing dentine for less than one third of surfaceILoss of enamel just exposing dentineCDefect less than 1 mm deep3B/L/OLoss of enamel exposing dentine for more than one third of surfaceILoss of enamel and substantial loss of dentineCDefect less than 1–2 mm deep4B/L/OComplete enamel loss–pulp exposure–secondary dentine exposureIPulp exposure or exposure of secondary dentineCDefect more than 2 mm deep–pulp exposure–secondary dentine exposure
This index was the first one designed to measure and monitor multifactorial tooth wear; a further pioneering feature was the ability to distinguish acceptable and pathological levels of wear, by comparison with threshold normal values for the age groups studied. Tooth wear was defined as pathological if the teeth became so worn that they do not function effectively or seriously mar the appearance—before they are lost through other causes—or the patient dies. Results from inter- and intra-reproducibility exercises were within a range regarded as acceptable for epidemiological purposes, and the index appears simple to use clinically—intra-orally or from models and photographs. However, some problems have been identified with the TWI, including the time necessary to apply to a whole dentition, amount of data generated and the comparisons with threshold levels for each age group; the thresholds proposed were high, erring towards understatement rather than exaggerations of pathological wear. Full use of the index as a research tool is not feasible without computer assistance.
A sign of professional confidence in this index is its adopted use by a number of different investigators in the UK looking at tooth wear prevalence and severity [3, 20, 25, 27], aetiology and risk [1, 21]. Other researchers have used it with modifications pertaining to the particular age group being studied. Millward et al. [18] made adjustments to study erosion in the primary and secondary dentitions, excluding cervical surfaces. Subjects were then grouped together to produce three classifications: no or mild erosion—no score >1, moderate—at least one tooth in dentition score 2, severe—at least one tooth in dentition score 3 or 4.
In a survey of elderly people, Steele et al. [31] combined low wear scores representing small losses of enamel and used the worst surface score per tooth as an overall tooth score. Donachie and Walls [7, 8] outlined various flaws in the tooth wear index as an epidemiological tool in the ageing population and suggested a need to increase the sensitivity of TWI at extremes of tooth wear, to take account of the capacity of the elderly to have adequate function in cases of significant wear. They suggested altering threshold values, amplifying scoring criteria and creating a sixth point to distinguish between exposure of secondary dentine and frank pulp exposure.
Development of indices
Many other indices have been proposed for measuring erosive tooth wear [2, 14, 15, 17, 23] which have their roots in the indices of Eccles [10] and Smith and Knight [30]. Linkosalo and Markkanen [15] utilised a qualitative index with listed diagnostic criteria to confirm lesions as erosive and a four-scale grading of severity, relating to involvement of dentine. Their scoring system was modified by Lussi et al. [17] to create an erosive index that has been used widely by European workers to score the facial, lingual and occlusal surfaces of all teeth except the third molars (Table 3).
Table 3Erosion index according to Lussi [16, 17]SurfaceScoreCriteriaFacial0No erosion. Surface with a smooth, silky glazed appearance, possible absence of developmental ridges1Loss of surface enamel. Intact enamel cervical to the erosive lesion; concavity on enamel where breadth clearly exceeds depth, thus distinguishing it from toothbrush abrasion. Undulating borders of the lesion are possible and dentine is not involved2Involvement of dentine for less than half of tooth surface3Involvement of dentine for more than half of tooth surfaceOcclusal/lingual0No erosion. Surface with a smooth, silky glazed appearance, possible absence of developmental ridges1Slight erosion, rounded cusps, edges of restorations rising above the level of adjacent tooth surface, grooves on occlusal aspects. Loss of surface enamel. Dentine is not involved2Severe erosions, more pronounced signs than in grade 1. Dentine is involved
O’Sullivan [23] proposed a new index for the measurement of erosion specifically in children. The index was qualitative with a broad attempt at quantification noting whether less or more than half of the surface was affected. Every tooth was examined and assigned a three-digit score relating to the site of erosion, severity (grade 0–5) and area of surface affected. O’Brien [22] reported the use of a partial recording system for measuring erosion in children in UK children’s dental health surveys, where only the facial and lingual surfaces of the primary and permanent maxillary incisor teeth were scored for erosion. Again, the criteria were mostly qualitative and descriptive, with a broad attempt made to quantify the area involved.
Bardsley et al. [2] pioneered a new, simplified version of TWI [30] when carrying out epidemiological studies on large numbers of adolescents in North West England (Table 4). Tooth wear scoring was essentially dichotomised into the presence or absence of dentine, with even cupping of dentine scoring one. A partial recording system was used, collecting data from 40 surfaces including occlusal surfaces of the four first molar teeth and the labial, incisal and lingual–palatal surfaces of the six upper and lower anterior teeth.
Table 4Simplified scoring criteria for TWI [2]ScoreCriteria0No wear into dentine1Dentine just visible (including cupping) or dentine exposed for less than 1/3 of surface2Dentine exposure greater than 1/3 of surface3Exposure of pulp or secondary dentine
However, despite calibration and training, difficulties were experienced diagnosing dentine exposure in the epidemiological field and there is some debate as to the significance of dentinal cupping when exposed dentine does not relate to significant amounts of tissue loss [12]. In a recent study of Ganss et al. [12], teeth were visually and histologically examined for presence of exposed dentine, and the correlation in accuracy between the two examinations was poor. The diagnosis of exposed dentine is obviously important but may not be accurate from visual examination alone.
Oilo et al. [24] concentrated on a different type of scoring system, with criteria based on treatment need. They criticised the use of indices that used a nonlinear scoring method, claiming calculated mean wear scores can be misleading. Their index was based on Ryge and Snyder’s [28] system for evaluating the quality of restorations and had five categories divided into two broad camps; Romeo, Sierra and Mike were satisfactory, whilst Tango and Victor indicated unacceptable levels of wear requiring treatment. All groups except Romeo were subdivided according to degree of dentine exposed and clinical findings such as pain, sensitivity and fracture of restorations, giving the impression of a cumbersome system. Dahl et al. [6] modified it with the introduction of even more categories, with an aim to establish subjective dental criteria for present and future evaluations of tooth wear and the need for treatment. In practice, these indices require experience for reliable use; individuals with differing clinical backgrounds will not get consistent, objective results.
Indices that attempt to visualise, measure and monitor the amount of worn enamel or exposed dentine by difficult direct clinical interpretation [13] and indirectly on accurate, serial study casts which must be retained [5, 11, 29, 32] have been described. Larsen et al. [14] recommended a new clinical index based on a combination of clinical examination, photographs and study casts, with complicated qualitative and quantitative criteria. Plaque-free teeth were clinically examined and photographed prior to taking silicone impressions for epoxy resin casts. They considered clinical and photographic data to be supplemental with final wear classification based on visual inspection of casts at ×10 magnification. Each tooth surface was scored, with six grades of erosion severity modelled from Smith and Knight [30]; the index and its criteria are complicated and time consuming.
Conclusion
Review of the literature on indices for tooth wear (or erosion) is confusing; there are too many indices proposed and used, with lack of standardisation in terminology. There are many epidemiological studies reported, but it is difficult to quantify the increases in prevalence reported internationally, as results are not easily comparable. It is doubtful whether any of the indices described are sensitive enough to monitor all but the most severe changes in tooth wear clinically and these cannot be used to measure a rate of wear [19]. It is a challenge to try to develop a simple index that can be used clinically to assess progression of wear.
To date, there is not one ideal index that can be used for epidemiological prevalence studies, clinical staging and monitoring, and it may be necessary to accept that one simple index does not yet exist to meet all requirements of both clinical and research teams. There should, however, be an aim for indices that can be relevant to both fields and can be used internationally in order to strengthen knowledge of dental erosion.


Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCα, PKCβ1, PKCγ, and PKCε are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.
1
Introduction
Phosphoinoisitide dependent kinase l (PDK1) phosphorylates a key threonine residue within the catalytic domain of a number of AGC family kinases, including diacylglycerol-regulated kinases of the protein kinase C (PKC) superfamily and phosphatidyl inositol-3 kinase (PI3K)-controlled serine kinases such as Akt/PKB and the 70-kilodalton ribosomal S6 kinase 1 (S6K1) [1]. PDK1-mediated phosphorylation of many of these AGC kinases is essential for their activation. Accordingly, deletion of PDK1 by homologous recombination removes one of the rate-limiting regulators of multiple serine kinases and causes embryonic lethality [2]. To circumvent this prenatal death and explore the role of PDK1 in different tissues, mice expressing PDK1 alleles flanked with the loxP Cre excision sequence (PDK1flΔneo/flΔneo mice) have been generated. These were first used to achieve muscle specific deletion of PDK1, producing mice that die early with severe defects in cardiac function [3]. The importance of PDK1 for T lymphocyte biology has also been explored by looking at T cell development following T lineage specific deletion of PDK1. These studies showed that deletion of PDK1 in T cell progenitors in the thymus results in a block in T cell differentiation, indicating that PDK1 has essential functions in T lymphocyte development [4].
The importance of PDK1 for T cell development in the thymus focuses attention on the identification of the PDK1 substrates directly involved in mediating the unique and non-redundant functions of this kinase in vivo. In this context, studies of mouse embryonic stem cells in which both PDK1 alleles have been deleted have shown that PDK1 is necessary for T-loop phosphorylation, and catalytic activity, of the AGC kinases Akt/PKB, S6K1 and p90 ribosomal S6 kinase (p90RSK) [5]. However, PDK1 is not required for the phosphorylation and activation of other AGC kinases such as PKA or AMPK [5].
One very important subfamily of AGC kinases that can be regulated by PDK1 are the protein kinase C enzymes. There are multiple isoforms of PKC: the classical PKCs (α, βI, βII and γ) which are regulated by calcium, diacylglycerol (DAG) and phospholipids; novel PKCs (δ, ε, η and θ) which are regulated by DAG and phospholipids; and the atypical PKCs (ζ and λ) which are insensitive to both calcium and DAG. The catalytic domain of PKCs have a conserved T-loop motif that is a substrate for PDK1, and it has been proposed that PDK1 is responsible for T-loop phosphorylation of the various PKC isoforms [6–8]. PDK1-mediated T-loop phosphorylation of PKCs is proposed to stabilize protein expression by acting as a priming signal that enables PKCs to achieve catalytic maturity [6,7]. Here, PDK1 docks to a hydrophobic motif in the C-terminus of newly synthesised PKC enzymes, enabling it to phosphorylate their T-loop sites. Following T-loop phosphorylation, PDK1 disengages enabling PKCs to autophosphorylate their C-terminal hydrophobic motifs and achieve catalytic maturity [9–11]. Once mature, PKCs remain inactive due to autoinhibition from their pseudosubstrate motifs and are only activated following the generation of second messengers, such as diacylglycerol and calcium.
The release of PDK1 from PKCs is rate limiting and so PDK1 is responsible for controlling the amount of mature/catalytically competent PKCs expressed in cells [11]. Hence, in the absence of PDK1, intracellular levels of PKCα, PKCβ1, PKCγ, and PKCε are strikingly reduced [6,12]. PKCξ levels are similar in wild-type and PDK1-null embryonic stem cell (ES cells) although PKCξ is not phosphorylated on its T-loop Thr410 site in the absence of PDK1 [6,13]. PKCδ expression is also reduced in PDK1-null cells [6,12] although there was still phosphorylation of the PKCδ T-loop site Thr505 in the residual pool of PKCδ still present in PDK1-null cells [13]. Moreover, there is some evidence in the literature that T-loop phosphorylation is not absolutely critical for PKCδ catalytic activity, since a bacterially-expressed Thr505A PKCδ mutant protein retains some activity in vitro [14]. Of note, a glutamic acid residue at position 500 within the PKCδ activation loop, which is important for catalytic activity [15], could potentially compensate for the lack of PKCδ T-loop phosphorylation in PDK1-null cells.
These studies support the hypothesis that PDK1 has an important role in regulating PKC phosphorylation and stability. However, it has not been shown unequivocally that the reduced levels of PKCs in PDK1-null cells abrogate PKC-mediated cellular responses. In this context, it has been shown that in the absence of PDK1 there is loss of Akt/PKB and S6K1 T-loop phosphorylation and a corresponding inability of cells to regulate phosphorylation of Akt/PKB or S6K1 substrates [5]. There is thus confidence that Akt/PKB and S6K1 function is defective in PDK1-null cells. This has not been determined for the PKCs because although PDK1-null cells have reduced endogenous PKC levels the integrity of downstream PKC effector pathways in PDK1-null cells has not been determined.
One well characterised target for novel PKCs is the serine kinase protein kinase D (PKD). Activation of PKD requires the phosphorylation of two key serine residues within the catalytic domain [16,17]. These residues are absolutely conserved through evolution from C. elegans to man and are substrates for novel PKCs. The activation of PKCs is thus required for PKD activation in a variety of cell lineages in response to a number of different stimuli [16,17]. For example, it has been demonstrated that PKD activity in lymphocytes is regulated by PKC-mediated phosphorylation of its catalytic domain [18,19] and phosphorylation of the PKD catalytic domain is reduced in PKCβ-deficient spleenocytes [20]. It would therefore be expected that PKD would remain inactive if PKCs cannot achieve catalytic maturity in PDK1-null cells. In support of this hypothesis, previous work has established that co-expression of PDK1 and the novel PKCε isoform, together with PKD, enhances PKD activity [21]. The hypothesis that PKD activation is dependent on PDK1 has not been fully investigated and is important to address as it is one way to assess whether there is residual functional PKC activity in PDK1-null cells. Accordingly, the objective of the present study was to examine whether loss of PDK1 results in loss of the PKC signalling pathway that regulates PKD.
2
Results and discussion
2.1
Activation of protein kinase D in PDK1−/− thymocytes
To investigate the impact of PDK1 loss on protein kinase D activation in PDK1 deficient T lymphocytes, mice expressing PDK1 alleles flanked with the loxP Cre excision sequence (PDK1flΔneo/flΔneo) were bred with transgenic mice expressing Cre recombinase under the control of the proximal p56lck promoter which induces expression of Cre in T cell progenitors in the thymus. It has been shown previously that in LckCre+PDK1flΔneo/flΔneo mice PDK1 is deleted in pre-T cells, preventing normal T cell development [4]. To explore the role of PDK1 in the regulation of PKD activity, wild-type or PDK1−/− pre-T cells were isolated and left unstimulated, stimulated with a crosslinking α-CD3 antibody (to activate preTCR signalling) or were stimulated with the phorbol ester PdBu (a diacylglycerol mimetic which activates classical/novel PKCs), as indicated. PKD catalytic activity was monitored using an antisera that recognises PKD molecules that are autophosphorylated on Ser916 [22]. The data (Fig. 1) show that in quiescent wild-type thymocytes PKD is not autophosphorylated on Ser916 whereas α-CD3 or PdBu stimulation strongly induced PKD catalytic activity, as shown by increased Ser916 autophosphorylation. The results also show that both α-CD3 and PdBu treatment induce strong PKD Ser916 autophosphorylation in PDK1−/− pre-T cells (Fig. 1). PKD activation is dependent on PKC-mediated phosphorylation of two key serine residues (Ser744/S748) within the PKD catalytic domain and as shown in Fig. 1, PKD is phosphorylated on Ser744/S748 following α-CD3 or PdBu activation in both wild-type and PDK1−/− pre-T cells.
The phosphorylation and activation of PKD in PDK1−/− pre-T cells argues that these cells still contain a functional, catalytically competent PKC(s) that is able to respond to diacylglycerol/phorbol ester signals. RNAi experiments have demonstrated that PKCε in Swiss 3T3 cells or PKCδ in HeLa cells are the PKC isoforms responsible for serine phosphorylation (Ser744/S748) within the PKD activation loop [23,24]. PKCδ has been shown to be residually phosphorylated on its T-loop Thr505 site in PDK1-null ES cells [13]. We therefore examined the phosphorylation status of the PKCδ T-loop site, Thr505, in PDK1−/− pre-T cells. The data in Fig. 1 show basal phosphorylation of PKCδ Thr505 in wild-type pre-T cells and also show that significant phosphorylation of PKCδ on Thr505 in retained in PDK1−/− pre-T cells. In contrast, as previously demonstrated, PDK1−/− pre-T cells have lost activity of the PKB/S6K1 signalling pathway that controls the phosphorylation of the ribosomal S6 subunit on Ser235/236 [4,25]. The data in Fig. 1 also explore the impact of PDK1 loss on the T-loop phosphorylation of p90RSK1 and PRK1. The data show Western blot analysis of wild type and PDK1 null pre-T cells with a phospho-specific antibody against Ser227 in p90RSK1 and show that this site is basally phosphorylated in wild-type pre-T cells but is not phosphorylated in PDK1-null pre-T cells. Similarly, basal phosphorylation of PRK1 on its Thr774 T-loop site is substantially reduced in PDK1-null pre-T cells. Thus, PDK1-null pre-T cells show defective activation of some, but not all, AGC family kinases: regulation of a PKC–PKD signalling pathway remains intact in these cells.
2.2
Activation of protein kinase D in PDK1−/− ES cells
The experiments in Fig. 1 show that PDK1 is not essential for PKD activation in thymocytes. To probe further the involvement of PDK1 in the regulation of PKC–PKD signalling, experiments in wild-type and PDK1−/− mouse embryonic stem cells were performed. Western blot analysis of wild-type and PDK1−/− ES cells with a PKCδ Thr505 phosphospecific antisera confirm that PKCδ Thr505 phosphorylation is detectable, albeit slightly reduced, in PDK1-null versus wild-type ES cells (Fig. 2A–C). To probe PKD activity, cells were left unstimulated or activated by exposure to serum or phorbol ester as indicated and PKD catalytic activity monitored using the pSer916 antisera. In quiescent, wild-type ES cells, PKD was not autophosphorylated on Ser916 but both serum and PdBu stimulation strongly and rapidly induced PKD activity as measured by increased Ser916 autophosphorylation (Fig. 2A and B). Serum or PdBu stimulation also induced PKD activity in the PDK1−/− ES cells (Fig. 2A and B) whereas p90RSK1 T-loop phosphorylation (Ser227) was basally detectable in wild-type ES cells but not in the PDK1−/− ES cells (Fig. 3). S6K1 signalling was also abrogated, as expected, in the PDK1−/− ES cells, as shown by the lack of phosphorylation of its substrate, the S6 ribosomal protein subunit (Fig. 2B).
The ability of serum and phorbol esters to activate PKD in PDK1-null ES cells is an indication that there is residual, catalytically competent PKC in these cells. PKC-mediated activation of PKD can be prevented by the PKC inhibitor GF109203X [17]. Therefore, the effects of GF109203X, on PKD activation in wild-type or PDK1−/− ES cells was examined. The data (Fig. 3) show that PKD was phosphorylated on Ser916 in activated PDK1+/+ and PDK1−/− cells but that PKD autophosphorylation on Ser916 was prevented by prior treatment of both cell types with GF109203X.
3
Concluding remarks
Loss of PDK1 in ES cells causes a reduction in levels of PKCα, βI, γ, δ, and ε [6,12] but it was not resolved whether the residual PKC in these cells is functional. As discussed above, there is a large body of evidence that PKD activation is mediated by novel PKCs such as PKCδ and ε and it has previously been proposed that PDK1 is required for activation of PKD [21], reflecting the requirement of PDK1 for phosphorylation of the key priming T-loop phosphorylation sites in PKCs. However, the present study shows that the hypothesis that PKD activation is dependent on PDK1 is incorrect. The reason this model fails is that there is not a complete loss of PKC activity in the absence of PDK1. We show that in PDK1-null pre-T cells PKCδ is still phosphorylated on its T-loop site Thr505; hence activation of pre-T cells with phorbol esters can still induce phosphorylation of PKD on its PKC substrate sequences Ser744/S748, inducing PKD activation and subsequent autophosphorylation on Ser916. In addition, the present data show that PKD can also be activated by phorbol esters or serum stimulation of PDK1-null ES cells in a PKC-regulated manner. Thus, the present results showing activation of PKD in PDK1−/− cells is evidence that at least one PKC-mediated signalling pathway is intact in the absence of PDK1.
While loss of PDK1 does not prevent the activation of PKD, the issue of whether the efficiency of PKD activation is affected in PDK1-null cells requires further experimentation. While robust activation of PKD signalling is observed in PDK1-null ES cells and pre-T cells in response to phorbol esters or CD3 crosslinking we cannot exclude the possibility that PKD activation may be compromised in PDK1-null mature peripheral T cells during physiological peptide:MHC stimulation, however these experiments await the development of genetic models to test this.
In summary, PDK1 is essential for pre-T cell development and its loss is associated with loss of PKB/S6K1-mediated signal transduction [4,25]. The present results now show loss of p90RSK and PRK1 T-loop phosphorylation in PDK1−/− pre-T cells but reveal that the PKCδ T-loop site Thr505 is still phosphorylated in PDK1-null pre-T cells. It has been proposed that the Thr505 T-loop site in PKCδ (as well as the corresponding T-loop site in PKCθ, [26]) can be autophosphorylated, which may explain why PKCδ Thr505 is still phosphorylated in the PDK1-null cells. Alternatively the present results could argue for the existence of another PKCδ priming kinase in developing thymocytes. Whatever the molecular basis for the continued phosphorylation of PKCδ following the loss of PDK1, the present results show that there is still residual functional PKC activity once PDK1 is lost. Hence, the importance of PDK1 for T cell development must reflect the requirement for PDK1 mediated phosphorylation of other members of the AGC serine kinase family, such as Akt/PKB, S6K1 or p90RSK, since phosphorylation and activity of these kinases is strictly dependent on PDK1 expression.
4
Materials and methods
4.1
Cell preparation and stimulation
PDK1+/+ and PDK1−/− murine embryonic stem cells [5] were grown on gelatinised tissue culture plates in Knockout DMEM supplemented with 10% knockout serum replacement, 15% foetal bovine serum (Hyclone), 0.1 mM non-essential amino acids, antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin and 1 μg/ml ciprofloxacin [Bayer Pharmaceuticals]), 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol and 25 ng/ml murine leukaemia inhibitory factor (LIF).
4.2
Mice
All mice used were between 5 and 7 weeks of age. Mice were bred and maintained in specific pathogen-free conditions and animal experimentation was approved by Home Office project license PPL60/3116. T-PDK1−/− mice were generated by crossing mice with PDK1 floxed alleles (PDK1flΔneo/flΔneo) with mice expressing Cre recombinase under the control of the p56lck promoter to ablate expression of PDK1 in T cells [2,4]. Control mice used for analyses of T-PDK1−/− mice were age-matched phenotypically normal littermates.
4.3
Purification of thymocyte subpopulations
Antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin were obtained from PharMingen. Cells were stained for surface expression of the following markers: CD4, CD8, CD25, CD44, CD3ε, γδ, B220, and Thy1. The DN4 subpopulation were defined by their cell surface marker expression and subsequently sorted using a Vantage cell sorter (Becton Dickinson) or AutoMacs magnetic cell sorter (Miltenyi Biotec).
4.4
Cell lysis and Western blot analysis
Cells were lysed for 10 min at 4 °C (20 × 106 cells/ml) using buffer: 100 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 20 mM sodium fluoride, 20 mM iodoacetamide, 2 μM EDTA, 1 mM sodium orthovanadate, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml chymostatin, 2 μg/ml antipain, 40 mM β-glycerophosphate and 1 mM phenylmethylsulfonylflouride. Soluble proteins were concentrated by precipitation with 1.5 vol. of acetone and separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to polyvinylidene difluoride membranes and detected by Western blot analysis with the indicated antibodies (Cell Signalling Technology).

pdk1, 3′-phosphoinositide-dependent protein kinase-1

s6k1, 70-kilodalton ribosomal s6 kinase 1


Testicular Dysgenesis Syndrome and the Estrogen Hypothesis: A Quantitative Meta-Analysis

Background Male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting from the disruption of gonadal development during fetal life, the testicular dysgenesis syndrome (TDS). The hypothesis that in utero exposure to estrogenic agents could induce these disorders was first proposed in 1993. The only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago, and other systematic reviews of the association between estrogenic compounds, other than the potent pharmaceutical estrogen diethylstilbestrol (DES), and TDS end points have remained inconclusive.
Impaired spermatogenesis, male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common underlying syndrome with a common aetiology resulting from the disruption of embryonic programming and gonadal development during fetal life, termed the testicular dysgenesis syndrome (TDS) (Sharpe and Skakkebaek 2003; Skakkebaek et al. 2001). A hormonal etiology most likely underlies this syndrome, although it is believed to have more than one cause, possibly including other than endocrine disruption. Some common causes of endocrine disruption include infection, diet and body weight, lifestyle, genetics, and environmental exposure, but endocrine-disrupting chemicals (EDCs), particularly those with estrogen-like properties, have received the most scientific attention.
The synthetic estrogenic drug diethylstilbestrol (DES) was prescribed to more than 5 million pregnant women from the late 1940s to the early 1970s to prevent abortions and pregnancy-related complications (Palmlund et al. 1993). Evidence later showed that maternal ingestion of DES during early pregnancy increased the risk of vaginal clear cell adenocarcinoma in female offspring (Herbst et al. 1971) and resulted in an increased incidence of malformations of the testes, the development of epididymal cysts, and impaired sperm quality in male offspring (Bibbo et al. 1977). During pregnancy, maternal estrogen levels are significantly elevated. However, more than 90% of maternal endogenous estrogens are effectively sequestered via binding to sex hormone binding globulin (SHBG), and thus the fetus is relatively protected (Joffe 2001; Vidaeff and Sever 2005). On the other hand, DES and ethinylestradiol do not bind well to SHBG, having a higher biopotency if ingested (Sharpe and Skakkebaek 2003; Vidaeff and Sever 2005). Additionally, transgenerational exposure is also possible when lipophilic xenoestrogens are mobilized during pregnancy and lactation (Colborn et al. 1993).
Previous systematic reviews of studies in which pregnant women were exposed to estrogens other than DES have failed to find evidence of an increased risk of urogenital abnormalities in the male offspring (Raman-Wilms et al. 1995; Storgaard et al. 2006; Toppari et al. 1996; Vidaeff and Sever 2005), and have raised the possibility that nonestrogenic or atypical estrogenic effects of DES exposure in utero induce male reproductive abnormalities. However, none of the effects of DES exposure on either male or female offspring of exposed wild-type pregnant mice were induced when administered to ERKO (ER-α knockout) mice (Couse et al. 2001), strongly suggesting an ER-α–mediated mechanism. There is, however, a body of experimental data that is consistent with an effect of antiandrogenic industrial chemicals on male sexual differentiation (Gray et al. 1999, 2000). Moreover, mechanisms other than endocrine disruption may be involved in testicular toxicity; for example, the nematocide dibromochloropropane, an alkylating agent, is one of the most potent known testicular toxins in adults (Joffe 2001). In this review we focus on the estrogen hypothesis of TDS.
Although several systematic reviews of the literature on the association between estrogenic agents and the disorders thought to belong to the TDS have been published, they are predominantly qualitative and the only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago (Toppari et al. 1996). The primary objective of a quantitative meta-analysis is to combine the results of previous studies examining a specific research question to arrive at a summary conclusion about a body of research. It has been found particularly useful when individual studies are too small to yield a valid conclusion, but it cannot, however, correct for bias and confounding. When applied to observational studies, subset analysis can be a useful tool to explore the reasons for discrepancies among the results of different studies.
The objectives of this research were therefore to carry out a quantitative meta-analysis of the association between three of the end points related to TDS and prenatal exposure to estrogenic agents that would account for both the size and quality of the studies included and yield updated summary estimates in light of the body of research carried out since the formulation of the estrogen hypothesis. Inclusion in this analysis was based on mechanistic criteria, and the plausibility of an ER-α–mediated mode of action was specifically explored. Moreover, subset analysis has been applied to categories of compounds with estrogenic potencies differing by several orders of magnitude in an attempt to detect the existence of any potency–response trend. Most of the studies of sperm quantity or quality have been concerned with time trends rather than etiology, and this end point was not considered further here.
Material and Methods
Identification and selection of literature
A computerized search was conducted using the databases PubMed (National Center for Biotechnology Information 2007) and Web of Science (ISI Web of Knowledge 2007) for the period 1970 to April 2007. The general search keywords were “estrogen,” “risk,” “dose,” and either “hypospadias,” “cryptorchidism,” or “testicular cancer.” A preliminary identification was performed by screening the titles and, if relevant, the abstracts of retrieved literature. The next stage was to check the citations and references of selected studies. This was an iterative process, repeated until no new study could be identified. A set of both inclusion and exclusion criteria was defined, and all relevant literature was then checked for eligibility. The inclusion criteria considered were a) study design, namely, either a case–control, cohort, or clinical trial; b) written in English; c) exposure to one or a mixture of known estrogenic compounds; and d) sufficient data reported to be used in meta-analysis.
The following exclusion criteria were used:
Exposure to a group of compounds (suspected endocrine disruptors) for which mode of action was unspecified, for example, pesticides.
Studies of exposure to phytoestrogens. Some phytoestrogens have been found to have a greater binding affinity for ER-β than for ER-α and can result in agonistic or antagonistic effects (Mueller et al. 2004).
Studies of maternal endogenous hormones.
Studies of the same cohort as this would bias the results towards the particular studies.
Incomplete data.
Data extraction and quality rating
In addition to the number of exposed and nonexposed cases and controls, and risk ratios (RRs) with their confidence intervals (CIs), information regarding the study design, estrogenic agent, geographic location of the study, and year of publication were extracted from the selected literature to allow subset analysis to be carried out. When more than one RR was reported, the following priorities were set for choice:
Adjusted RRs were used, except when the study provided only unadjusted estimates.
When multiple estimates were given, the RR estimator on which the authors had relied for their assessment of causal association was used.
Overall RRs were chosen instead of those derived from further stratifications. If an overall estimate was not provided, the RRs of the maximum duration of exposure or the maximum exposure concentration were chosen.
Several aspects of the quality of each study were also recorded according to a rating scheme adapted from those previously described (Altman 1991; Rushton 2000). Every criterion was assessed on a scale of 0 to 2, 0 suggesting that it was not present, 1 when it was unclear, and 2 when that criterion was satisfied. A maximum score of 50 and 52 could be assigned for retrospective (case–control) and prospective (cohort and clinical trials) studies, respectively. This enabled a quality sensitivity analysis to be performed to check the influence of studies with low quality on the pooled estimate.
Data analysis
Graphical representation
The RRs and CIs were plotted against the year of publication to determine whether any positive or negative trends in reporting RRs had occurred over time. Similarly, quality scores were plotted against the year of publication to investigate whether the quality of studies improved over time. To assess publication bias, a funnel plot (SE vs. RR) was produced based on the assumption that smaller studies are less precise in their RRs and thus have less weight and larger SE and should scatter more widely at the lower end of the graph, whereas larger studies will tend to be closer together (Sterne et al. 2001). Forest plots present the RRs against the reference of the study and help check homogeneity visually.
Statistical pooling
Pooled estimates and 95% CIs were calculated using both a fixed-effects model (Mantel–Haenszel method) and a random-effects model (DerSimonian–Laird method), allowing evaluation of the dependence of the conclusions of the analysis on the model assumptions. A summary estimate is considered statistically significant at the 0.05 level if its CI does not include unity.
The Mantel–Haenszel pooled effect estimate was used in a chi-square statistical test of homogeneity to assess the between-study variance. The magnitude of the test statistics depends on the weight of each study. When the number of studies is low or the studies themselves are small, the test statistic Q tends to be small. Tests of heterogeneity in meta-analyses are generally low in their power to reject the null hypothesis of homogeneity. For this reason, the chi-square statistical test of homogeneity was carried out at both 0.05 and 0.1 significance levels. Additionally, pooled estimates calculated using fixed effect and random effect models differ only if there is lack of homogeneity between studies. The estimates obtained by both methods were therefore compared to better assess potential heterogeneity between studies, in which case a single summary estimate of effect may be considered inappropriate.
Subset and sensitivity analyses
To investigate potential sources of heterogeneity between studies, we performed subset analyses for the study design, estrogenic agent, and geographic location.
Some studies exploring the influence of hormonal treatment during pregnancy did not specify the type of hormone. From what is known of the hormonal treatment of common conditions occurring during pregnancy, it was deemed reasonable to assume that they would have been likely to include estrogens, and these studies were included in the analysis. The validity of this assumption was tested by applying stricter criteria and calculating a summary estimate of effect excluding any study in which the hormone used had not been specified. Further sensitivity analysis was performed by excluding low-quality studies and extremes (exclusion of the studies with the largest and smallest RR estimators and exclusion of the studies with the largest and smallest weights) to verify that either the quality of the studies or one particular study did not have an excessive influence on the pooled estimate.
Results
A total of 50 studies were identified for the association between in utero exposure to estrogenic agents and hypospadias and/or cryptorchidism, including 16 that had not been included in previous systematic reviews. Sixteen studies, of which 8 were new studies, were included in the calculation of a summary estimate of effect for either or both end points (Table 1). Studies predating the formulation of the TDS hypothesis often were designed to explore the association of in utero exposure to a range of pharmaceuticals with birth malformations. Other than 2 recent studies for which pesticide exposure was determined by chemical analysis of specific compounds, assessment of exposure to pesticides is generally derived from the occupation of the mother and specific agents are not identified.
Of the 12 studies identified for the association with testicular cancer, only 3 were excluded from the calculation of a summary estimate of effect (Table 2).
Hypospadias
The data from studies included in the meta-analysis for hypospadias are summarized in Table 3. Three extreme values, two greater than and one lower than unity, can be identified visually from the forest plot of the RRs and their CIs (Figure 1). These extremes correspond to studies with larger SEs, and the shape given to the funnel plot (Figure 2) by those smaller positive studies would be consistent with publication bias. These two extreme positive risk ratios were, however, reported after what is commonly referred to as “third-generation exposure” to DES, when the mother herself had been exposed to DES prenatally. It was recognized that the inclusion of such studies in the meta-analysis could have introduced heterogeneity, and the influence of this choice was investigated in the subset analysis. Plots of the quality score and RRs versus year of publication did not suggest any significant trends in quality of the studies or estimates of effect over time (not shown).
The pooled estimates of effect by both the Mantel–Haenszel and DerSimonian–Laird methods are very close to unity, and no relationship between in utero exposure to estrogenic agents and hypospadias could be detected (Table 4). None of the chi-square tests allowed the rejection of the null hypothesis of homogeneity between the studies at the 0.05 or 0.1 level of statistical significance. The subsets of studies in which exposure to DES and pharmaceutical estrogens were investigated yielded statistically significant risk ratios with both models, although the modest discrepancy between the fixed-effects and random-effects estimates suggests heterogeneity. Summary estimates for the latter subset were no longer significant when studies that included DES exposure were excluded. Although these results were based on four studies that all addressed in utero exposure to oral contraceptives, some heterogeneity between studies remained. Excluding the studies of third-generation exposure to DES, values for the summary estimate of effect were found to be 1.33 (95% CI, 0.63–2.83) by the Mantel–Haenszel method and 1.31 (95% CI, 0.52–3.26) by the DerSimonian–Laird method, a very modest and nonsignificant increase in risk. Excluding third-generation exposure from the DES subset yielded estimates of 2.02 (95% CI, 1.12–3.65) by the Mantel–Haenszel method and 2.00 (95% CI, 0.97–4.15) by the DerSimonian–Laird method, on the basis of two studies investigating exposure to any estrogenic drug during the first trimester of pregnancy. The difference between the results obtained by the two models for studies of third-generation exposure to DES was reduced only slightly by excluding the study by Klip et al. (2002); the Mantel–Haenszel method yielded an estimate of 2.46 (95% CI, 0.91–6.67) and the DerSimonian–Laird method of 2.18 (95% CI, 0.64–7.46). The latter study’s cohort had been recruited in a fertility clinic, and whether results obtained with subfertile women are generalizable to all women exposed to DES in utero has been questioned (Hernandez-Diaz 2002).
Although the equality of the results obtained by both methods for the environmental estrogens subset suggests those results are robust, the influence of the weight of the study by Vrijheid et al. (2003) cannot be underestimated, as shown by the sensitivity analysis. Exclusion of this study from the analysis yielded a statistically significant Mantel–Haenszel estimate but a lower and not statistically significant DerSimonian–Laird estimate, revealing heterogeneity. A statistically significant estimate was obtained for prospective studies by the Mantel–Haenszel method, but the wide difference with the estimate using the random effect model was suggestive of heterogeneity. Geographic subsets point to a higher risk in Latin America, although the pooled estimates for this location were based on only two studies and did not reach statistical significance.
In addition to the results of the sensitivity analysis presented in Table 4, a pooled estimate of effect was calculated when a stricter inclusion criterion was applied, namely, excluding results from the study by Monteleone-Neto et al. (1981). This had little influence on the overall result, generating summary estimates of 0.97 (95% CI, 0.83–1.13) for the fixed effect model or 0.93 (95% CI, 0.80–1.09) for the random effect model.
Cryptorchidism
Data for the six studies included in the meta-analysis for cryptorchidism can be found in Table 5. The results of only two studies significantly differ from unity, as illustrated by the forest plot (Figure 3). The small number of eligible studies renders analysis of the funnel plot and potential for publication bias difficult (Figure 4). The SEs do, however, illustrate well that the studies were all relatively small. No time trends for the estimate of effect or the quality of studies could be detected (not shown).
As presented in Table 6, the pooled estimates of effect by both the Mantel–Haenszel and DerSimonian–Laird methods are marginally superior to unity, and their relative divergence implies there may be sources of heterogeneity. Chi-square tests did not, however, detect that any of the subsets were significantly heterogeneous. Excluding studies in which DES exposure was examined, either exclusively or along with hormonal therapeutics, yielded summary estimates consistent with no relationship. Statistical pooling of the studies including DES exposure generated a statistically significant estimate by the Mantel–Haenszel method, suggesting a doubling of the risk of cryptorchidism after in utero exposure to DES. The same estimate by the DerSimonian–Laird method did not, however, reach statistical significance and the difference relative to the fixed effect model is indicative of heterogeneity. The heterogeneity introduced by the DES subset of studies can again be observed by comparing the results obtained for all pharmaceutical estrogens with those obtained by pooling the two studies of accidental use of oral contraceptives during pregnancy. Study design also appeared to be a source of heterogeneity. If case–control studies are prone to recall bias, this subset also included the study with the highest estimate, itself a source of heterogeneity, as shown by the sensitivity analysis. Excluding the study by Depue (1988) reduced the difference between estimates by both models, the Mantel–Haenszel estimate then calculated as 1.29 (95% CI, 0.87–1.91) and that by the DerSimonian–Laird method as 1.23 (95% CI, 0.81–1.86). This was also observed for the American subset of studies. When the Depue (1988) study is omitted, the Mantel–Haenszel method yielded a no longer statistically significant estimate of 1.34 (95% CI, 0.84–2.14) and the DerSimonian–Laird method an estimate of 1.27 (95% CI, 0.72–2.23).
Applying a stricter exclusion criterion to studies examining hormonal treatment did not affect which studies were included in the meta-analysis of cryptorchidism. The study with the highest weight appears to lower the overall estimates, whereas increasing quality seems to reduce heterogeneity and lower the estimate of effect toward unity. These variations did not, however, influence the overall conclusion that aside from the DES studies subset, summary estimates did not detect any association between in utero exposure to estrogenic substances and cryptorchidism.
Testicular cancer
Nine studies were included in the meta-analysis of testicular cancer and the data used are summarized in Table 7. Of these, 4 had not been included in the summary estimate previously calculated by Toppari et al. (1996). The lack of homogeneity between studies is evident from the forest plot (Figure 5). Further, the funnel plot (Figure 6) also illustrates the relatively small size of the included studies. Although a positive trend over time was found for the quality of the included studies (Figure 7), no significant time trend could be detected for the effect size (not shown).
Both the fixed and random effect models yield a statistically significant estimate; however, the discrepancy between the two results is suggestive of heterogeneity despite the result from the chi-square test (Table 8). Conversely, the subset analysis was limited by the similarity of the question addressed by the studies included. Eight of the nine studies were interested in hormonal exposure and were conducted in the United States. Despite this, statistically significant heterogeneity between the studies was detected at the 0.1 level. Pooling the two studies examining DES exposure specifically produced a raised but statistically nonsignificant result. Despite the unexplained heterogeneity, all estimates that were calculated point to a doubling of the risk of developing testicular cancer after exposure to estrogenic agents in utero. The work on chlorinated biphenyls (PCBs) by Hardell et al. (2004) was the only study examining environmental estrogens. Its size was relatively small, and it did not detect such an effect.
Applying a stricter exclusion criterion to studies examining hormonal treatment excluded four studies from the meta-analysis; namely, Brown et al. (1986), Gershman and Stolley (1988), Henderson et al. (1979), and Weir et al. (2000). This resulted in a slightly lower Mantel–Haenszel estimate of 1.98 (95% CI, 1.23–3.18) and if the DerSimonian–Laird estimate remained equal to 1.59, because of the wider confidence interval (95% CI, 0.93–2.69), statistical significance was no longer achieved. The sensitivity analysis is consistent with some heterogeneity between the studies, the estimates obtained being relatively sensitive to the exclusion of particular studies varying above and below a risk estimate of 2. The quality of the studies seemed to explain at least some of this heterogeneity.
Discussion
While it is clear that hypospadias, cryptorchidism, and testicular cancer are all positively associated with prenatal exposure to DES, this meta-analysis was unable to produce evidence that such effects were associated with environmental estrogens or even accidental use of oral contraceptives during pregnancy. This is consistent with the results obtained in earlier meta-analyses (Raman-Wilms et al. 1995; Toppari et al. 1996).
The main limitations of meta-analysis are a) the susceptibility of its summary results to publication bias, b) the influence of the quality of studies, c) the possibility of including multiple results from the same study, and finally, d) heterogeneity between studies that could lead to invalid conclusions. The methodology employed in this present review attempts to address these issues. Additionally, the importance of carrying out and reporting a sensitivity analysis was illustrated by the case of hypospadias where the weight attributed to one particularly large study had a nonnegligible influence on the results. In this particular case, the study by Vrijheid et al. (2003) inferred exposure to phthalates from registry data about occupation, and although such an approach can allow the analysis of a great number of cases, assessment of exposure is much more likely to be prone to confounding. The number of studies included in meta-analyses lies typically between 5 and 15, and the results presented here also fall within this range. The size of the homogeneity test statistic depends on both the number and size of individual studies. The funnel plots offer a good visual representation of the precision and size of individual studies, and it is clear that most studies published on the association between estrogenic agents and the probable end points of a TDS were found to be relatively small. The chi-square tests had, therefore, a relatively low power to detect heterogeneity. However, in the absence of statistical heterogeneity, the results of the fixed effect and random effect models should be virtually identical, and the comparison of results obtained by applying both the Mantel–Haenszel and DerSimonian–Laird models enabled the exploration of sources of heterogeneity despite this low statistical power.
If the quality of the studies was found to explain some of the heterogeneity observed, particularly in the case of testicular cancer, the remaining heterogeneity could not be explained solely by the fact that environmental, and therefore generally much weaker, estrogens were included in the analysis. The systematic review of published literature yielded relatively few studies examining the association of male urogenital abnormalities or testicular cancer with environmental estrogens specifically; a number of studies concerned with an association with broad categories of putative endocrine disruptor, most often pesticides, were excluded from the meta-analyses. This illustrates the difficulties associated with assessment of exposure, pesticide exposure often being inferred from parental occupation rather than direct measurement. Furthermore, there is increasing evidence that, in accordance with pharmacokinetic theory, the effects of xenobiotics acting via the same mechanism can be predicted fairly accurately by concentration addition (Zhu et al. 2006). Accurately accounting for combined exposure or adjusting for the confounding introduced by environmental exposures will probably require the development of mechanism-specific biomarkers of exposure.
When DES is excluded, there is no conclusive evidence of an effect of pharmaceutical estrogens. Exposure to such estrogens is related mainly to the accidental use of oral contraceptives during pregnancy or hormonal pregnancy tests. Such estrogenic pharmaceuticals often are given in combination with progestagens, and it is legitimate to question whether unopposed estrogens would have the same effects as opposed estrogens. This also highlights another difficulty associated with exposure assessment, that of critically sensitive periods of development and the ascertainment of whether exposure took place during a “window” of susceptibility to hormone disruption. Nonetheless, studies in which maternal levels of hormones were measured in the first and third trimester of pregnancy do not support an association with elevated estrogen levels but rather indicate that a lower estrogen/androgen ratio and/or higher levels of α-fetoproteins may be beneficial (McGlynn et al. 2005; Zhang et al. 2005). If in animals both estrogenic and antiandrogenic compounds have been associated with end points consistent with those of human TDS (Fisher 2004; Veeramachaneni 2000), epidemiologic evidence remains elusive. Alternatively, the doubling of the risk estimates of all three effects associated with DES exposure would be consistent with a shared etiology and the TDS hypothesis. It does not constitute conclusive evidence of an estrogenic mode of action, however, as common etiologic factors could be related to the underlying condition for which DES was prescribed. Furthermore hypospadias, cryptorchidism, and testicular cancer have all been found to be associated with low birth weight, suggesting a potential association with an underlying placental defect.
The understanding of the importance of endogenous estrogens in normal adult testicular function is becoming clearer. Their roles during fetal life, however, remain relatively unclear, but those mediated by the ER-αor ER-β have been shown to differ (Habert et al. 2006). Interestingly, DES has been found to have similar affinity for both receptors, whereas estradiol has only a slightly stronger affinity for ER-αcompared with ER-β (Mueller et al. 2004). ER-α has been detected in undifferentiated gonads as early as 10 days postconception in the mouse and found to be localized in the Leydig cells of fetal testis in rodents (Habert et al. 2006). Studies of the expression of ER-α and ER-β in human and nonhuman primates have so far yielded inconsistent results. Gaskell et al. (2003) reported that ER-α could not be detected in human fetal testes between weeks 12–19 of gestation, whereas Shapiro et al. (2005) found that ER-α was apparent by week 12, its levels peaked at 16 weeks before diminishing, and it was localized in Leydig cells. Current research focus has shifted to the role played by testosterone, anti-Müllerian hormone and insulin-like factor 3 produced by the fetal testes during masculinization. In the male rat, exposure to high levels of estrogens has been shown not only to suppress testosterone production but also to downregulate the expression of the androgen receptor protein in reproductive target tissues including the testes, Wolffian duct, and prostate (Sharpe 2006). Further research in this area may help shed light on possible mechanisms of injury or relevance of the rodent model.
The subset analyses did not generate many clues to explain the heterogeneity of the collected data. This is, however, consistent with the wide geographic variability in the incidence of the conditions of interest (Boisen et al. 2004; Richiardi et al. 2004). Interactions between genetic susceptibility and the environment have been the focus of research in this area (Martin et al. 2007), and advances in genomics have allowed the identification of polymorphisms associated with hypospadias, cryptorchidism, and testicular cancer (Beleza-Meireles et al. 2006; Kurahashi et al. 2005; Starr et al. 2005; Yoshida et al. 2005). Such discoveries may, however, give rise to as many questions as they offer to answer. This is well illustrated by the recent identification of the association of a variant of the gene for the ER-α with hypospadias and cryptorchidism in Japanese cohorts (Watanabe et al. 2007; Yoshida et al. 2005) that has now been found to be associated with a decreased incidence of hypospadias in a European cohort (Galan et al. 2007).
Conclusion
The modest increase in risk for all three end points associated with DES exposure is consistent with a shared etiology and the TDS hypothesis, whereas the results of the subset analyses suggest the existence of yet unidentified sources of heterogeneity between studies or within the study populations. Although 10 years of further research on the potential effects of endocrine disruptors on male reproductive health have provided some clues regarding the etiology and mechanism of conditions such as hypospadias, cryptorchidism, and testicular cancer, there is still no conclusive evidence of the role played by environmental estrogens.

testicular dysgenesis
estrogen
meta-analysis
hypospadias
cryptorchidism
testicular cancer
diethylstilbestrol
endocrine disruption
environment
oral contraceptives