Local production of 17ß-oestradiol in the endometrium during the implantation window: a pilot study.

Abstract: Sex steroids are converted to bioactive metabolites and vice versa by endometrial steroid-metabolising enzymes. Studies indicate that alterations in this metabolism might affect endometrial receptivity. This pilot study determined whether the endometrial formation and inactivation of 17ß-oestradiol differed between the supposedly embryo-receptive endometrium and non-receptive endometrium of women undergoing IVF/intracytoplasmic sperm injection (ICSI). Endometrial biopsies were obtained from IVF/ICSI patients 5-8 days after ovulation in a natural cycle, prior to their second IVF/ICSI cycle with fresh embryo transfer (ET). Endometrial biopsies from patients who achieved clinical pregnancy after fresh ET (n = 15) were compared with endometrial biopsies from patients that did not conceive after fresh ET (n = 15). Formation of 17ß-oestradiol (oxidative 17ß-hydroxysteroid dehydrogenases (HSDs)), oestrone (reductive HSD17Bs) and inhibition of HSD17B1 activity were determined by high-performance liquid chromatography. The endometrial transcriptome was profiled using RNA sequencing followed by principal component analysis and differentially expressed gene analysis. The false discovery rate-adjusted P < 0.05 and log fold change >0.5 were selected as the screening threshold. Formation and inactivation of 17ß-oestradiol resulted similar between groups. Inhibition of HSD17B1 activity was significantly higher in the non-pregnant group when only primary infertile women (n = 12) were considered (27.1%, n = 5 vs 16.2%, n = 7, P = 0.04). Gene expression analysis confirmed the presence of HSD17B1 (encoding HSD17B1), HSD17B2 (encoding HSD17B2) and 33 of 46 analysed steroid metabolising enzymes in the endometrium. In the primary infertile subgroup (n = 10) 12 DEGs were found including LINC02349 which has been linked to implantation. However, the exact relationship between steroid-metabolising enzyme activity, expression and implantation outcome requires further investigation in larger, well-defined patient groups. Lay summary: Sex hormones are produced and broken down by enzymes that can be found in the endometrium (the inner lining of the womb). This enzyme activity might influence the chances of becoming pregnant. We compared (i) enzyme activity in the endometrium of 15 women who did and 15 women who did not become pregnant in their second in vitro fertilisation attempt, (ii) how enzyme activity can be blocked by an inhibitor, and (iii) differences in gene expression (the process by which instructions in our DNA are converted into a product). Enzyme activity was similar between groups. We found that in women who have never been pregnant in the past, inhibition of enzyme activity was higher and found differences in a gene that has been linked to the implantation of the embryo, but future studies should be performed in larger, well-defined patient groups to confirm these findings.

Economic evaluation of endometrial scratching before the second IVF/ICSI treatment: a cost-effectiveness analysis of a randomized controlled trial (SCRaTCH trial).

STUDY QUESTION: Is a single endometrial scratch prior to the second fresh IVF/ICSI treatment cost-effective compared to no scratch, when evaluated over a 12-month follow-up period? SUMMARY ANSWER: The incremental cost-effectiveness ratio (ICER) for an endometrial scratch was €6524 per additional live birth, but due to uncertainty regarding the increase in live birth rate this has to be interpreted with caution. WHAT IS KNOWN ALREADY: Endometrial scratching is thought to improve the chances of success in couples with previously failed embryo implantation in IVF/ICSI treatment. It has been widely implemented in daily practice, despite the lack of conclusive evidence of its effectiveness and without investigating whether scratching allows for a cost-effective method to reduce the number of IVF/ICSI cycles needed to achieve a live birth. STUDY DESIGN, SIZE, DURATION: This economic evaluation is based on a multicentre randomized controlled trial carried out in the Netherlands (SCRaTCH trial) that compared a single scratch prior to the second IVF/ICSI treatment with no scratch in couples with a failed full first IVF/ICSI cycle. Follow-up was 12 months after randomization.Economic evaluation was performed from a healthcare and societal perspective by taking both direct medical costs and lost productivity costs into account. It was performed for the primary outcome of biochemical pregnancy leading to live birth after 12 months of follow-up as well as the secondary outcome of live birth after the second fresh IVF/ICSI treatment (i.e. the first after randomization). To allow for worldwide interpretation of the data, cost level scenario analysis and sensitivity analysis was performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: From January 2016 until July 2018, 933 women with a failed first IVF/ICSI cycle were included in the trial. Data on treatment and pregnancy were recorded up until 12 months after randomization, and the resulting live birth outcomes (even if after 12 months) were also recorded.Total costs were calculated for the second fresh IVF/ICSI treatment and for the full 12 month period for each participant. We included costs of all treatments, medication, complications and lost productivity costs. Cost-effectiveness analysis was carried out by calculating ICERs for scratch compared to control. Bootstrap resampling was used to estimate the uncertainty around cost and effect differences and ICERs. In the sensitivity and scenario analyses, various unit costs for a single scratch were introduced, amongst them, unit costs as they apply for the United Kingdom (UK). MAIN RESULTS AND THE ROLE OF CHANCE: More live births occurred in the scratch group, but this also came with increased costs over a 12-month period. The estimated chance of a live birth after 12 months of follow-up was 44.1% in the scratch group compared to 39.3% in the control group (risk difference 4.8%, 95% CI -1.6% to +11.2%). The mean costs were on average €283 (95% CI: -€299 to €810) higher in the scratch group so that the point average ICER was €5846 per additional live birth. The ICER estimate was surrounded with a high level of uncertainty, as indicated by the fact that the cost-effectiveness acceptability curve (CEAC) showed that there is an 80% chance that endometrial scratching is cost-effective if society is willing to pay ∼€17 500 for each additional live birth. LIMITATIONS, REASONS FOR CAUTION: There was a high uncertainty surrounding the effects, mainly in the clinical effect, i.e. the difference in the chance of live birth, which meant that a single straightforward conclusion could not be ascertained as for now. WIDER IMPLICATIONS OF THE FINDINGS: This is the first formal cost-effectiveness analysis of endometrial scratching in women undergoing IVF/ICSI treatment. The results presented in this manuscript cannot provide a clear-cut expenditure for one additional birth, but they do allow for estimating costs per additional live birth in different scenarios once the clinical effectiveness of scratching is known. As the SCRaTCH trial was the only trial with a follow-up of 12 months, it allows for the most complete estimation of costs to date. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by ZonMW, the Dutch organization for funding healthcare research. A.E.P.C., F.J.M.B., E.R.G. and C.B. L. reported having received fees or grants during, but outside of, this trial. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL5193/NTR 5342).

Endometrial scratching in women with one failed IVF/ICSI cycle-outcomes of a randomised controlled trial (SCRaTCH).

STUDY QUESTION: Does endometrial scratching in women with one failed IVF/ICSI treatment affect the chance of a live birth of the subsequent fresh IVF/ICSI cycle? SUMMARY ANSWER: In this study, 4.6% more live births were observed in the scratch group, with a likely certainty range between -0.7% and +9.9%. WHAT IS KNOWN ALREADY: Since the first suggestion that endometrial scratching might improve embryo implantation during IVF/ICSI, many clinical trials have been conducted. However, due to limitations in sample size and study quality, it remains unclear whether endometrial scratching improves IVF/ICSI outcomes. STUDY DESIGN, SIZE, DURATION: The SCRaTCH trial was a non-blinded randomised controlled trial in women with one unsuccessful IVF/ICSI cycle and assessed whether a single endometrial scratch using an endometrial biopsy catheter would lead to a higher live birth rate after the subsequent IVF/ICSI treatment compared to no scratch. The study took place in 8 academic and 24 general hospitals. Participants were randomised between January 2016 and July 2018 by a web-based randomisation programme. Secondary outcomes included cumulative 12-month ongoing pregnancy leading to live birth rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with one previous failed IVF/ICSI treatment and planning a second fresh IVF/ICSI treatment were eligible. In total, 933 participants out of 1065 eligibles were included (participation rate 88%). MAIN RESULTS AND THE ROLE OF CHANCE: After the fresh transfer, 4.6% more live births were observed in the scratch compared to control group (110/465 versus 88/461, respectively, risk ratio (RR) 1.24 [95% CI 0.96-1.59]). These data are consistent with a true difference of between -0.7% and +9.9% (95% CI), indicating that while the largest proportion of the 95% CI is positive, scratching could have no or even a small negative effect. Biochemical pregnancy loss and miscarriage rate did not differ between the two groups: in the scratch group 27/153 biochemical pregnancy losses and 14/126 miscarriages occurred, while this was 19/130 and 17/111 for the control group (RR 1.21 (95% CI 0.71-2.07) and RR 0.73 (95% CI 0.38-1.40), respectively). After 12 months of follow-up, 5.1% more live births were observed in the scratch group (202/467 versus 178/466), of which the true difference most likely lies between -1.2% and +11.4% (95% CI). LIMITATIONS, REASONS FOR CAUTION: This study was not blinded. Knowledge of allocation may have been an incentive for participants allocated to the scratch group to continue treatment in situations where they may otherwise have cancelled or stopped. In addition, this study was powered to detect a difference in live birth rate of 9%. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study are an incentive for further assessment of the efficacy and clinical implications of endometrial scratching. If a true effect exists, it may be smaller than previously anticipated or may be limited to specific groups of women undergoing IVF/ICSI. Studying this will require larger sample sizes, which will be provided by the ongoing international individual participant data-analysis (PROSPERO CRD42017079120). At present, endometrial scratching should not be performed outside of clinical trials. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by ZonMW, the Dutch organisation for funding healthcare research. J.S.E. Laven reports grants and personal fees from AnshLabs (Webster, Tx, USA), Ferring (Hoofddorp, The Netherlands) and Ministry of Health (CIBG, The Hague, The Netherlands) outside the submitted work. A.E.P. Cantineau reports 'other' from Ferring BV, personal fees from Up to date Hyperthecosis, 'other' from Theramex BV, outside the submitted work. E.R. Groenewoud reports grants from Titus Health Care during the conduct of the study. A.M. van Heusden reports personal fees from Merck Serono, personal fees from Ferring, personal fees from Goodlife, outside the submitted work. F.J.M. Broekmans reports personal fees as Member of the external advisory board for Ferring BV, The Netherlands, personal fees as Member of the external advisory board for Merck Serono, The Netherlands, personal fees as Member of the external advisory for Gedeon Richter, Belgium, personal fees from Educational activities for Ferring BV, The Netherlands, grants from Research support grant Merck Serono, grants from Research support grant Ferring, personal fees from Advisory and consultancy work Roche, outside the submitted work. C.B. Lambalk reports grants from Ferring, grants from Merck, grants from Guerbet, outside the submitted work. TRIAL REGISTRATION NUMBER: Registered in the Netherlands Trial Register (NL5193/NTR 5342). TRIAL REGISTRATION DATE: 31 July 2015. DATE OF FIRST PATIENT'S ENROLMENT: 26 January 2016.

Endometrial scratching in women with implantation failure after a first IVF/ICSI cycle; does it lead to a higher live birth rate? The SCRaTCH study: a randomized controlled trial (NTR 5342).

BACKGROUND: Success rates of assisted reproductive techniques (ART) are approximately 30%, with the most important limiting factor being embryo implantation. Mechanical endometrial injury, also called 'scratching', has been proposed to positively affect the chance of implantation after embryo transfer, but the currently available evidence is not yet conclusive. The primary aim of this study is to determine the effect of endometrial scratching prior to a second fresh in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle on live birth rates in women with a failed first IVF/ICSI cycle. METHOD: Multicenter randomized controlled trial in Dutch academic and non-academic hospitals. A total of 900 women will be included of whom half will undergo an endometrial scratch in the luteal phase of the cycle prior to controlled ovarian hyperstimulation using an endometrial biopsy catheter. The primary endpoint is the live birth rate after the 2nd fresh IVF/ICSI cycle. Secondary endpoints are costs, cumulative live birth rate (after the full 2nd IVF/ICSI cycle and over 12 months of follow-up); clinical and ongoing pregnancy rate; multiple pregnancy rate; miscarriage rate and endometrial tissue parameters associated with implantation failure. DISCUSSION: Multiple studies have been performed to investigate the effect of endometrial scratching on live birth rates in women undergoing IVF/ICSI cycles. Due to heterogeneity in both the method and population being scratched, it remains unclear which group of women will benefit from the procedure. The SCRaTCH trial proposed here aims to investigate the effect of endometrial scratching prior to controlled ovarian hyperstimulation in a large group of women undergoing a second IVF/ICSI cycle. TRIAL REGISTRATION: NTR 5342 , registered July 31st, 2015. PROTOCOL VERSION: Version 4.10, January 4th, 2017.

Molecular aspects of implantation failure.

Despite expanding global experience with advanced reproductive technologies, the majority of IVF attempts do not result in a successful pregnancy, foremost as a result of implantation failure. The process of embryo implantation, a remarkably dynamic and precisely controlled molecular and cellular event, appears inefficient in humans and is poorly understood. However, insights gained from clinical implantation failure, early pregnancy loss, and emerging techologies that enable molecular interrogation of endometrial-embryo interactions are unravelling this major limiting step in human reproduction. We review current molecular concepts thought to underlie implantation failure, consider the contribution of embryonic and endometrial factors, and discuss the clinical value of putative markers of impaired endometrial receptivity. Finally we highlight the nature of the dialogue between the maternal endometrium and the implanting embryo and discuss the concept of natural embryo selection. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Recognition of heat shock protein 60 epitopes in children with type 1 diabetes.

BACKGROUND: Treatment with a specific HSP60 epitope in new onset of type 1 diabetes (T1D) patients has been shown to preserve endogenous insulin production. Previously, recognition of pan HLA-DR-binding HSP60 epitopes in various autoimmune diseases was found; this study investigated recognition of these epitopes in newly diagnosed T1D patients and correlated findings to the occurrence of a partial remission. METHODS: Peripheral blood mononuclear cells of 18 children with T1D were prospectively collected at disease onset and a few months after diagnosis. Epitope-specific T-cell proliferation and cytokine production (intracellular and in culture supernatants) were measured. Results were compared with 31 longstanding T1D patients and ten healthy controls. RESULTS: Although HSP60 epitope-specific T-cell proliferative responses were detected, overall proliferative responses were low. At onset, epitope-specific intracellular IFN-γ production was higher in T1D patients compared with healthy controls (p < 0.05). At follow-up, both IL-10 and IFN-γ production were higher in those without a partial remission than in those with a partial remission (both p < 0.05). Also, IL-10 and IFN-γ production were higher compared with onset for patients without a PR (both p < 0.01). In supernatants of HSP60 epitope-specific T-cell cultures, no substantial differences in cytokine production were found between T1D patients with and without a partial remission, either at onset or a few months after onset. As patient numbers were small, results should be interpreted with caution. CONCLUSIONS: Pan-DR-binding HSP60 peptides induced low peptide-specific proliferative responses and peptide-specific production of some, mainly intracellular, cytokines in T1D patients. Recognition did not differ significantly between patient groups and various time points.

The molecular basis of recurrent pregnancy loss: impaired natural embryo selection.

Recurrent pregnancy loss (RPL) is a common and distressing disorder. Chromosomal errors in the embryo are the single most common cause, whereas uterine factors are invariably invoked to explain non-chromosomal miscarriages. These uterine factors are, however, poorly defined. The ability of a conceptus to implant in the endometrium is normally restricted to a few days in the menstrual cycle. A limited 'window of implantation' ensures coordinated embryonic and endometrial development, thereby minimizing the risk of late implantation of compromised embryos. In this paper, we review emerging evidence, indicating that RPL is associated with impaired differentiation of endometrial stromal cells into specialized decidual cells. From a functional perspective, this differentiation process, termed decidualization, is not only critical for placental development but also signals the end of the implantation window and bestows on the endometrium the ability to recognize, respond to and eliminate implanting compromised embryos. Thus, we propose that spontaneous decidualization of the human endometrium, which inevitably causes menstrual shedding in the absence of a viable conceptus, serves as functional 'window for natural embryo selection'. Conversely, impaired decidualization predisposes to late implantation, negates embryo quality control and causes early placental failure, regardless of the embryonic karyotype. This pathological pathway also explains the common observation that many RPL patients seem exceptionally fertile, often conceiving within one or two cycles. Thus, as the clinical correlate of inappropriate uterine receptivity, 'superfertility' should be considered as a genuine reproductive disorder that requires targeted intervention.

Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity.

Investigation of human embryo implantation requires a non-disruptive means of studying the endometrium during the window of implantation. This study describes a novel approach of cytokine profiling in endometrial secretions. Endometrial secretions aspirated prior to embryo transfer from 210 women undergoing IVF or intracytoplasmic sperm injection were analysed by a multiplex immunoassay. Ten mediators [interleukin (IL)-1beta, IL-6, IL-12, IL-18, tumour necrosis factor-alpha, macrophage migration inhibitory factor, eotaxin, monocyte chemotactic protein-1, interferon-gamma inducible protein-10, vascular endothelial growth factor] were detectable in 90-100% of the samples. Heparin-binding epidermal growth factor, IL-5, IL-17, IL-10, Dickkopf homologue-1 and IL-15 were detected in 23-76%, whereas interferon-gamma was not detectable in any of the samples. To assess possible contamination of samples, cervical mucus was also aspirated for comparative analysis in 22 women. The endometrial cytokine profile differed significantly from cervical mucus. Pregnancy rates of the study participants who underwent endometrial secretion aspiration were compared with 210 controls matched for important prognostic variables; no significant differences were found. In conclusion, cytokine profiling in endometrial secretion offers an objective, non-disruptive means of analysing the in-vivo milieu encountered by the embryo and offers a new and potentially valuable approach to studying the endometrial factor in human embryo implantation.