A comprehensive MRI investigation to identify potential biomarkers of Osgood Schlatter disease in adolescents: A cross sectional study comparing Osgood Schlatter disease with controls.

BACKGROUND: Osgood-Schlatter disease (OSD) is the most common knee pain complaint among adolescents playing sports. Despite this, there remains controversy over the pathophysiology and whether specific anatomical characteristics are associated with OSD. PURPOSE: This study aimed to systematically and comprehensively characterize adolescents with OSD using magnetic resonance imaging (MRI) compared to pain-free controls, including both tissue abnormalities that may be associated with OSD, as well as anatomical characteristics. A secondary objective was to identify potential imaging biomarkers associated with pain. STUDY DESIGN: Cross-sectional study. METHODS: Adolescents with OSD and controls were recruited from 2020 to 2022. Following a clinical exam, demographics, pain, sports participation, and Tanner stage were collected. Knee MRI was conducted on the participants' most symptomatic knee (OSD) or the dominant leg (controls). RESULTS: Sixty-seven adolescents (46 with OSD and 30 controls) were included. 80% of participants with OSD had at least one tissue alteration compared to 54% of controls. Compared to controls, OSD had 36.3 (95%CI 4.5 to 289.7) higher odds of bony oedema at the tibial tuberosity, and 32.7 (95%CI 4.1 to 260.6) and 5.3 (95%CI 0.6 to 46.2) higher odds of bony oedema at the  tibial epiphysis and metaphysis respectively. Participants with OSD also had higher odds of fluid/oedema at the patellar tendon (12.3 95%CI 3.3 to 46.6), and superficial infrapatellar bursitis (7.2).  Participants with OSD had a more proximal tendon attachment (mean tibial attachment portion difference, -0.05, 95% CI: -0.1 to 0.0, p = 0.02), tendon thickness (proximal mean difference, -0.09, 95% CI: -0.4 to 0.2, p = 0.04; distal mean difference, -0.6, 95% CI: -0.9 to -0.2, p = 0.01). Those with bony/tendon oedema had 1.8 points (95% CI: 0.3 to 3.2) higher pain on palpation than those without (t = -2.5, df = 26.6, p = 0.019), but there was no difference between these groups in a functional single leg pain provocation. CONCLUSION: Adolescents with OSD present with tissue and structural abnormalities on MRI that differed from age-matched controls. The majority had findings in the patellar tendon and bone, which often co-occurred. However, a small proportion of OSD also presents without alterations. It appears these findings may be associated with clinical OSD-related pain on palpation of the tibial tuberosity. CLINICAL RELEVANCE: Our highlight the pathophysiology on imaging, which has implications for understanding the mechanism and treatment of OSD.

Fertility of frozen sex-sorted sperm at 4 × 106 sperm per dose in lactating dairy cows in seasonal-calving pasture-based herds.

The objective was to evaluate the reproductive performance of frozen sex-sorted sperm at 4 × 106 sperm per dose (SexedULTRA 4M, Sexing Technologies, Navasota, TX) relative to frozen conventional sperm in seasonal-calving pasture-based dairy cows. Semen from Holstein-Friesian (n = 8) and Jersey (n = 2) bulls was used. Four of the Holstein bulls used were resident at or near a sex-sorting laboratory (Cogent, UK, or ST Benelux, the Netherlands). The remaining 6 bulls were located at studs in Ireland. For these 6 bulls, ejaculates were collected, diluted with transport medium, and couriered to Cogent in parcel shippers. Transit time from ejaculation to arrival at the sorting laboratory was 6 to 7 h. For all bulls, ejaculates were split and processed to provide frozen conventional sperm (CONV) at 15 × 106 sperm per straw and frozen sex-sorted (SS) sperm at 4 × 106 sperm per straw and used to inseminate lactating dairy cows after spontaneous estrus. Pregnancy diagnosis was performed by ultrasound scanning (n = 7,246 records available for analysis). Generalized linear mixed models were used to examine effects on pregnancy per AI (P/AI) at first artificial insemination, with sperm treatment (CONV vs. SS), bull (n = 10), and treatment × bull interaction as the fixed effects, and herd (n = 142) as a random effect. Overall, P/AI was greater for cows inseminated with CONV than for those inseminated with SS (59.9% vs. 45.5%; 76.0% relative to CONV). This study was not designed to compare resident bulls vs. shipped ejaculates, but the magnitude of the difference between P/AI achieved by CONV and SS was apparently less for resident bulls (60.3% vs. 50.2%) than for shipped ejaculates (58.6% vs. 40.7%). We discovered a treatment × bull interaction for shipped ejaculates (P/AI ranged from 45 to 86% relative to CONV) but not for the resident bulls (P/AI ranged from 81 to 87% relative to CONV). Relative P/AI of SS compared with CONV was greater in cows with high or average fertility potential (76.1% and 78.3%, respectively) than in cows with low fertility potential (58.1%). In 33.1% of the enrolled herds, the P/AI achieved with SS was 90% or more of the P/AI achieved with CONV; this was mainly explained by herds in which SS performed exceptionally well but CONV performed poorly. In conclusion, SS had lower overall P/AI compared with CONV; however, P/AI achieved with SS was dependent on the bull, fertility potential of the cow, and herd. Strategies to improve the P/AI with SS in seasonal-calving pasture-based lactating dairy cows require further research.

Evaluation of delayed timing of artificial insemination with sex-sorted sperm on pregnancy per artificial insemination in seasonal-calving, pasture-based lactating dairy cows.

The objective was to use ovulation synchronization with timed artificial insemination (TAI) to evaluate the effect of timing of artificial insemination (AI) with frozen sex-sorted sperm on fertility performance in pasture-based compact calving herds. Ejaculates from 3 Holstein-Friesian bulls were split and processed to provide frozen sex-sorted sperm (SS) at 4 × 106 sperm per straw, and frozen conventional sperm at 15 × 106 sperm per straw (CONV). A modified Progesterone-Ovsynch protocol was used for estrous synchronization, with TAI occurring 16 h after the second GnRH injection for cows assigned to CONV, and either 16 h (SS-16) or 22 h (SS-22) for cows assigned to SS. Pregnancy diagnosis was conducted by transrectal ultrasound scanning of the uterus 35 to 40 d after TAI (n = 2,175 records available for analysis). Generalized linear mixed models were used to examine the effects of treatment on pregnancy per artificial insemination (P/AI). Fixed effects included treatment (n = 3), bull (n = 3), treatment by bull interaction, parity (n = 4), days-in-milk category (n = 3), and treatment by days-in-milk category, with herd (n = 24) included as a random effect. Pregnancy per AI was greater for CONV compared with both SS-16 and SS-22 (61.1%, 49.0%, and 51.3%, respectively), and the SS treatments did not differ from each other (relative P/AI for SS-16 and SS-22 vs. CONV were 80.2% and 84.0%, respectively). There were significant bull and treatment by bull interaction effects. Additional analysis was undertaken using a model that included herd as a fixed effect. This analysis identified marked herd-to-herd variation (within-herd relative P/AI for the combined SS treatments vs. CONV ranged from 48-121%). The tertile of herds with the best performance achieved a mean relative P/AI of 100% (range = 91-121%), indicating that P/AI equivalent to CONV is achievable with SS. Conversely, the tertile of herds with the poorest performance achieved a mean relative P/AI of 67% (range = 48-77%). We found that SS resulted in poorer overall P/AI compared with CONV sperm regardless of timing of AI. Marked variation existed between herds; however, one-third of herds achieved P/AI results equal to CONV. Identification of factors responsible for the large herd-to-herd variation in P/AI with SS, and development of strategies to reduce this variation, warrant further research.

Transparency in the eye region of an ostracod carapace ( Macrocypridina castanea, Myodocopida).

Many myodocopid ostracods are unusual in that they have well-developed compound eyes yet must view their environment through a shell. The cypridinid Macrocypridina castanea is relatively large among ostracods (about 5-10 mm) and is a pelagic predator. This species possess highly pigmented shells with a transparent region lying just above the eye. Here we examine the ultrastructure and transparency of this window using electron microscopy, serial-block face scanning electron microscopy and X-ray diffraction analysis and optical modelling. An internal, laminar stack was identified within the window region of the shell that formed a more regular half-wave reflector than in non-window regions, and where the distance between molecules in the chitin-protein fibrils decreases as compared to the non-window area. This results in excellent transmission properties-at around 99% transmission-for wavelengths between 350 and 630 nm due to its half-wave reflector organization. Therefore, blue light, common in the mid and deep sea, where this species inhabits, would be near-optimally transmitted as a consequence of the sub-micrometre structuring of the shell, thus optimizing the ostracod's vision. Further, pore canals were identified in the shell that may secrete substances to prevent microbial growth, and subsequently maintain transparency, on the shell surface. This article is part of the theme issue 'Bioinspired materials and surfaces for green science and technology'.

The relationship between anogenital distance and fertility, and genome-wide associations for anogenital distance in Irish Holstein-Friesian cows.

The evaluation of anogenital distance (AGD), the distance from the center of the anus to base of the clitoris, as a potential fertility trait for genetic selection in dairy cows has generated recent interest. The objectives of this cross-sectional observational study were to (1) characterize the distribution and variability of AGD, (2) determine factors associated with AGD, (3) estimate heritability for AGD, (4) identify single nucleotide polymorphisms (SNP) associated with phenotypic variation of AGD, and (5) validate the relationship between categories of AGD and fertility in Irish Holstein-Friesian cows. Anogenital distance was measured using digital calipers in 1,180 Holstein cows (mean ± standard deviation: 225 ± 79 d in milk) from 10 dairy herds located in Munster, Ireland. In addition, age (yr), weight (kg), height at hip (cm), and body condition score (BCS) at the time of AGD measurement were determined in a subset of 281 cows. Genotype information available from 908 cows was subsequently imputed to the Illumina Bovine High Density BeadChip (Illumina Inc., San Diego, CA) for genome-wide association analysis of phenotypic variation in AGD. Overall, AGD had a normal distribution and high variability (mean ± standard deviation; 119.2 ± 11.6 mm). Anogenital distance was weakly but positively associated with cow age, hip height, and body weight, and negatively associated with BCS; the phenotypic variation in AGD that was explainable by these variables was small (coefficient of determination; R2 = 0.09, 0.06, 0.10, and 0.02, respectively). The estimated heritability for AGD was 0.37 (standard error of mean ± 0.08). Six SNP of suggestive significance were identified on Bos taurus autosomes 6, 15, 20, and 26; however, none of these SNP was related to previously identified candidate genes for fertility. Cows were categorized into quartiles (Q1; 86 to 111 mm; n = 311, Q2; 112 to 120 mm; n = 330; Q3; 121 to 127 mm; n = 265, and Q4; 128 to 160 mm; n = 274) based on AGD and the association with reproductive outcomes examined (21-d submission rate, pregnancy to first AI, pregnancy rate within 21, 42 and 84-d after the farm mating start date, and number of times bred). None of the reproductive variables differed significantly between AGD categories. In summary, despite identification of high variability and moderate heritability for AGD in Irish Holstein-Friesian cows, reproductive outcomes did not differ between categories of AGD. This latter result differs from our previous finding of an inverse relationship between AGD and pregnancy outcomes in first- and second-parity Canadian Holstein cows, emphasizing the need to test and validate this new phenotype in diverse cow populations.

Prevalence, glucose control and relative survival of people with Type 2 diabetes in the UK from 1991 to 2013.

AIMS: To characterize the prevalence of Type 2 diabetes between 1991 and 2013 in the UK and to determine whether corresponding glucose control and survival had changed in the diabetic population during this period. METHODS: For this retrospective cohort study, people diagnosed with Type 2 diabetes between 1991 and 2013 were identified from the Clinical Practice Research Datalink (CPRD) and the annual point prevalence calculated. Mean HbA1c by year was estimated. The Cox proportional hazards model was used to calculate the risk of all-cause mortality by year for incident cases of Type 2 diabetes treated with glucose-lowering therapy. RESULTS: Crude prevalence of diagnosed Type 2 diabetes increased from 1.32% [95% confidence interval (95% CI) 1.30% to 1.34%] in 1991 to 4.54% (4.52% to 4.56%) in 2013. Mean HbA1c for people with diagnosed Type 2 diabetes was 71 mmol/mol (8.6%) in 1991, 59 mmol/mol (7.5%) in 2003 and 58 mmol/mol (7.5%) in 2013. For diagnosed Type 2 diabetes treated with glucose-lowering therapy, when compared with 1991, the hazard ratio for all-cause mortality was 0.33 (0.27-0.41) in 2013. CONCLUSION: The prevalence of diagnosed Type 2 diabetes trebled in the UK between 1991 and 2013. Improved survival in people with diagnosed Type 2 diabetes is likely to account, at least in part, for the increase in prevalence observed.

In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen-thawed stallion semen.

The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100×106 spermatozoa mL-1 with 0.02mM vitamin E (VE) and 0, 1, 10 or 20ng mL-1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120min and viability, acrosome integrity and membrane fluidity were evaluated at 30min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20×106 spermatozoa mL-1 and stored at 4°C. After 1, 24, 48 and 72h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function.

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

Two-dimensional knee valgus displacement as a predictor of patellofemoral pain in adolescent females.

Patellofemoral pain (PFP) is a prevalent lower limb musculo-skeletal injury in adolescent females. Female athletes with PFP display increased frontal plane knee joint motion in comparison to control subjects. The current investigation aimed to determine prospectively whether two-dimensional knee valgus displacement during landing could predict the risk of developing PFP. Seventy-six injury-free adolescent female athletes (age = 12.9 ±0.35 years) participated. At baseline participants performed three drop vertical jump trials from a 31-cm box. A standard video camera was used to record frontal plane knee joint kinematics. Over the 24-month follow-up, eight participants developed PFP, as diagnosed by a Chartered Physiotherapist. Knee valgus displacement was significantly increased in those who developed PFP compared to those who did not (mean difference = 7.79°; P = 0.002; partial eta squared = 0.07). Knee valgus displacement ≥10.6° predicted PFP with a sensitivity of 0.75 and specificity of 0.85. The associated positive likelihood ratio was 5. These results have clinical utility suggesting that two-dimensional analysis could be implemented to screen for increased risk of PFP in adolescent female athletes.

Glucose-lowering with exogenous insulin monotherapy in type 2 diabetes: dose association with all-cause mortality, cardiovascular events and cancer.

AIMS: To evaluate the association between insulin exposure and all-cause mortality, incident major adverse cardiovascular events (MACE) and incident cancer in people with type 2 diabetes treated with insulin monotherapy. METHODS: For this retrospective study, people with type 2 diabetes who progressed to insulin monotherapy from the year 2000 were identified from the UK Clinical Practice Research Datalink. The risks of progression to serious adverse outcomes were compared using Cox proportional hazards models. In the main analysis, insulin exposure was introduced into the model as prescribed international units per kilogram per day, as a cumulative, continuous, annually updated, time-dependent covariable. RESULTS: A total of 6484 subjects with type 2 diabetes who progressed to treatment with insulin monotherapy from the year 2000 onwards were followed for a mean of 3.3 years. The event numbers were as follows: deaths, n = 1110; incident MACE, n = 342; incident cancers, n = 382. Unadjusted event rates were 61.3 deaths per 1000 person-years, 26.4 incident MACE per 1000 person-years and 24.6 incident cancers per 1000 person-years. The adjusted hazard ratios in relation to 1-unit increases in insulin dose were 1.54 [95% confidence interval (CI) 1.32-1.78] for all-cause mortality, 1.37 (95% CI 1.05-1.81) for MACE and 1.35 (95% CI 1.04-1.75) for cancer. CONCLUSIONS: There was an association between increasing exogenous insulin dose and increased risk of all-cause mortality, MACE and cancer in people with type 2 diabetes. The limitations of observational studies mean that this should be further investigated using an interventional study design.

Mortality risk with sulphonylureas compared to metformin.

Current clinical guidelines in the USA and the UK recommend first-line glucose-lowering treatment with metformin monotherapy for glucose control in type 2 diabetes, where not contraindicated. Consequently, the proportion of people treated with sulphonylureas is decreasing. The purpose of this commentary is to discuss the risks and benefits associated with sulphonylurea monotherapy versus metformin monotherapy and the evidence that, in comparison with metformin, sulphonylureas cause increased harm to people with type 2 diabetes.

How many people inject insulin? UK estimates from 1991 to 2010.

AIMS: We set out to estimate the prevalence rate of insulin use in the UK population, the total number of people in the UK who use insulin, the proportion of users with type 1 and type 2 diabetes and changes between 1991 and 2010. METHODS: Patients receiving prescriptions for insulin were identified in the Clinical Practice Research Datalink and attributed a diagnosis of type 1 or type 2 diabetes. The annual prevalence of insulin use was calculated and applied to population data. RESULTS: The crude prevalence rate of insulin use increased from 2.43 (95% CI 2.38-2.49) per 1000 population in 1991 to 6.71 (6.64-6.77) per 1000 in 2010. The largest change was an increase in the prevalence of insulin users with a diagnosis of type 2 diabetes from 0.67 (0.64-0.70) to 4.34 (4.29-4.39) per 1000 population. The absolute number using insulin increased from 137 000 people (121 000-155 000) in 1991 to 421 000 (400 000-444 000) in 2010. The proportion taking insulin alone (as against combination with oral agents) decreased from 97% in the first decade to 37% in the second. CONCLUSION: The number of people using insulin trebled between 1991 and 2010, largely due to a considerable increase in the number of people with type 2 diabetes using insulin.

Association between first-line monotherapy with sulphonylurea versus metformin and risk of all-cause mortality and cardiovascular events: a retrospective, observational study.

AIMS: To evaluate the risk of all-cause mortality and major adverse cardiovascular events (MACE) for patients exposed to first-line monotherapy with sulphonylurea or metformin. METHODS: Data were from the Clinical Practice Research Datalink (CPRD). Patients with type 2 diabetes were selected if initiated with metformin or sulphonylurea monotherapy as their first-line glucose-lowering regimen 2000-2012. The primary endpoint was all-cause mortality; the secondary endpoint was MACE (myocardial infarction or stroke). Times to endpoints were compared using Cox proportional hazards models. Additional analyses were performed on subsets matched directly on key characteristics and by propensity score. RESULTS: In the main analysis, 76 811 patients were prescribed metformin monotherapy (mean follow-up 2.9 years) and 15 687 sulphonylurea monotherapy (mean follow-up 3.1 years). A total of 2604 patients were included in each arm of the directly matched cohorts and 8836 in the propensity-matched. With respect to all-cause mortality, using all three analytical approaches the hazard ratio (HR) was significantly increased for sulphonylurea compared with metformin: adjusted HR = 1.580 (95% CI 1.483-1.684) for the main analysis, 1.902 (1.733-2.088) for those matched on propensity score, and 1.272 (1.021-1.584) for the directly matched cohort analysis. For MACE, the respective HRs were 1.196 (1.090-1.313), 1.202 (1.001-1.442) and 0.814 (0.578-1.148), respectively. CONCLUSIONS: All-cause mortality was significantly increased in patients prescribed sulphonylurea compared with metformin monotherapy. Whilst residual confounding and confounding by indication may remain, this study indicates that first-line treatment with sulphonylurea monotherapy should be reconsidered.

Combination therapy with metformin plus sulphonylureas versus metformin plus DPP-4 inhibitors: association with major adverse cardiovascular events and all-cause mortality.

AIMS: To compare the risk of major adverse cardiovascular events (MACE) and mortality for combination therapies with metformin and either sulphonylurea (SU) or dipeptidyl peptidase-4 inhibitor (DPP-4i). METHODS: Data were from the UK Clinical Practice Research Datalink (CPRD). Patients with type 2 diabetes were selected if initiated with combination therapies comprising metformin plus SU or DPP-4i 2007-2012. The co-primary endpoints were all-cause mortality and MACE (myocardial infarction or stroke). Times to endpoints were compared using Cox proportional hazards models. Additional analyses were performed on subsets matched directly on key characteristics and by propensity score. RESULTS: A total of 33 983 patients were prescribed SU and 7864 DPP-4i, and 5447 patients in each cohort could be matched directly and 6901 by propensity score. In the main analysis, there were 716 MACE events and 1217 deaths. Crude event rates for MACE were 11.3 events per 1000 person-years (pkpy) for SU, versus 5.3 pkpy for DPP-4i. For all-cause mortality, rates were 16.9 versus 7.3 pkpy, respectively. Following adjustment, there was a significant increase in the adjusted hazard ratio (aHR) for all-cause mortality in those exposed to SU across all analytical models: aHR = 1.357 (95% CI 1.076-1.710) for all subjects, 1.850 (1.245-2.749) directly matched and 1.497 (1.092-2.052) propensity-matched. For MACE, aHR was 1.710 (1.280-2.285) for all subjects, 1.323 (0.832-2.105) directly matched and 1.547 (1.076-2.225) propensity-matched. CONCLUSIONS: There was a reduction in all-cause mortality for patients treated with metformin combined with DPP-4i versus metformin plus SU, and a similar trend for MACE.

Can people with type 2 diabetes live longer than those without? A comparison of mortality in people initiated with metformin or sulphonylurea monotherapy and matched, non-diabetic controls.

AIMS: Clinical and observational studies have shown an increased risk of cardiovascular events and death associated with sulphonylureas versus metformin. However, it has never been determined whether this was due to the beneficial effects of metformin or detrimental effects of sulphonylureas. The objective of this study was therefore to compare all-cause mortality in diabetic patients treated first-line with either sulphonylurea or metformin monotherapy with that in matched individuals without diabetes. METHODS: We used retrospective observational data from the UK Clinical Practice Research Datalink (CPRD) from 2000. Subjects with type 2 diabetes who progressed to first-line treatment with metformin or sulphonylurea monotherapy were selected and matched to people without diabetes. Progression to all-cause mortality was compared using parametric survival models that included a range of relevant co-variables. RESULTS: We identified 78,241 subjects treated with metformin, 12,222 treated with sulphonylurea, and 90,463 matched subjects without diabetes. This resulted in a total, censored follow-up period of 503,384 years. There were 7498 deaths in total, representing unadjusted mortality rates of 14.4 and 15.2, and 50.9 and 28.7 deaths per 1000 person-years for metformin monotherapy and their matched controls, and sulphonylurea monotherapy and their matched controls, respectively. With reference to observed survival in diabetic patients initiated with metformin monotherapy [survival time ratio (STR) = 1.0], adjusted median survival time was 15% lower (STR = 0.85, 95% CI 0.81-0.90) in matched individuals without diabetes and 38% lower (0.62, 0.58-0.66) in diabetic patients treated with sulphonylurea monotherapy. CONCLUSIONS: Patients with type 2 diabetes initiated with metformin monotherapy had longer survival than did matched, non-diabetic controls. Those treated with sulphonylurea had markedly reduced survival compared with both matched controls and those receiving metformin monotherapy. This supports the position of metformin as first-line therapy and implies that metformin may confer benefit in non-diabetes. Sulphonylurea remains a concern.

Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success.

STUDY QUESTION: Do human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity? SUMMARY ANSWER: Blastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not. WHAT IS KNOWN ALREADY: Human preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium. STUDY DESIGN, SIZE, DURATION: We used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3). MAIN RESULTS AND THE ROLE OF CHANCE: Blastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse. WIDER IMPLICATIONS OF THE FINDINGS: This new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success. STUDY FUNDING/COMPETING INTEREST: This study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.