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Study question Which clinical parameters predict a high oocyte maturation rate in patients undergoing IVF treatment? Summary answer Time between oocyte collection and insemination demonstrated significant association with oocyte maturation and represents a parameter that could be optimised in IVF cycles. What is known already Oocyte maturation is an important factor determining IVF outcomes and can be a rate-limiting step for patients undergoing treatment. A number of clinical and laboratory variables may affect this process, including the choice of trigger prior to oocyte collection, and certain laboratory procedures. Identification of which of these are predictors of maturation in individual centres enables local protocols to be optimised. Study design, size, duration This is a retrospective study of 714 oocyte collections from 661 women between January 2020 to November 2020 treated in a large, single centre in the UK. Subsequent fertilisation on fresh oocytes consisted of 371 IVF and 343 ICSI cycles. Participants/materials, setting, methods Patient and treatment data was collected by clinical staff at time of treatment. Either GnRH agonist, hCG or double trigger were administered 36 hours before collection. Prior to ICSI, oocyte maturation was assessed by visualisation of polar body (PB) extrusion. After IVF, the number of 2PNs plus unfertilised oocytes with PB extrusion were assessed. Univariate analyses consisted of Mann-Whitney test, t-test, Fisher’s Exact test or ANOVA. Potential predictors were investigated by logistic regression. Main results and the role of chance The end point was maturation rate, defined as high (greater or equal to 70%) or low (less than 70%). Factors predictive of a high rate included insemination more than 4 hours after collection. Oocytes inseminated over 4 hours post-collection displayed significantly higher maturation rates than oocytes inseminated less than 2 hours after collection (69% and 61% respectively; P = 0.01). Oocytes inseminated between 2–4 hours also had higher maturation than those inseminated less than 2 hours post-collection, but this did not reach significance (67% and 61%, respectively; P = 0.06). Further, oocytes fertilised by ICSI had significantly higher maturation than conventional IVF (77% and 67%, respectively, P Limitations, reasons for caution Varying means of data collection across clinics and between clinical staff inevitably leads to provision of incomplete data and should be taken into consideration alongside interpretation. Prescription bias of specific triggers to certain patient demographics should be noted. Wider implications of the findings: Collectively, these results suggest that greater time between oocyte collection and insemination could be recommended to IVF clinics that wish to optimise their oocyte maturation. Triggering final maturation with GnRH agonist versus hCG or dual trigger did not have a significant effect on oocyte maturation when adjusted for confounders. Trial registration number Not applicable

Study question Do established donor and recipient clinical markers predict recipient clinical pregnancy and live birth rates (LBRs) in a vitrified oocyte donation programme? Summary answer Recipient BMI and previous miscarriages predicted cumulative LBR. Likelihood of clinical pregnancy and LBR was higher in recipients of donors aged 23–29 than donors 18–22. What is known already The influence of age on ovarian reserve underlies the upper limit of 35 years for UK donors. However, recent evidence suggests that oocyte aneuploidy rates follow an inverse U-shaped curve in relation to a woman’s age. Conflicting evidence exists regarding the impact of other donor-related factors including BMI, AMH, oocyte yield and prior reproductive history on recipient outcomes. Moreover, the effect of recipient age, BMI, and reproductive history on oocyte donation outcome remains unclear. Study design, size, duration Retrospective cohort study of 325 altruistic oocyte donors matched to a total of 511 recipients. Only first donations taking place between January 2017 and December 2019 were included. Participants/materials, setting, methods All oocyte donors were altruistic volunteers aged 18–35 with no prior infertility diagnosis. Donor and recipient screening for suitability and safety was carried out according to the Human Fertilisation Embryology Authority guidelines. Backward stepwise logistic regression was used to identify donor, recipient and embryology parameters predictive of recipient primary outcomes defined as clinical pregnancy and live birth, either cumulative or after the first embryo transfer (ET). Main results and the role of chance A total of 705 fresh and frozen/thawed ETs were performed, of which 76% were elective single embryo transfers (eSETs) of blastocysts (96.5%), resulting in a cumulative clinical pregnancy and LBR of 83.5% and 70.5% respectively after 3 ETs. Recipient BMI and previous miscarriages were predictors of cumulative LBR (p Limitations, reasons for caution The present study included only healthy oocyte donors, thus conclusions may not apply to subfertile or less healthy women. Male factors were not accounted for. Wider implications of the findings: We demonstrate the efficacy of vitrified oocyte donation treatment and identify recipient BMI, previous miscarriages and embryology parameters as predictors of cumulative LBR. Additionally, the choice of donors aged 18–22 instead of older donors is found not to be advantageous for increasing the chance of clinical pregnancy and live birth. Trial registration number Not applicable

Study question Does reducing estradiol levels with letrozole co-treatment during ovarian stimulation with gonadotropins for IVF impact endocrinological and reproductive outcome markers in expected normal responders? Summary answer Letrozole co-treatment maintained follicular phase physiological serum estradiol levels, increased gonadotropin and androgen levels, and increased progesterone in the luteal phase. What is known already Ovarian stimulation for IVF causes supraphysiologic estradiol levels, which exert pituitary suppression reducing gonadotropin stimulation of the corpus luteum. Furthermore, stimulation may increase progesterone in the late follicular phase, reported to impair clinical outcomes, through a putative effect on endometrial maturation and embryo-endometrial asynchrony. Co-treatment with the highly selective aromatase inhibitor letrozole during ovarian stimulation has been shown to reduce estradiol levels and FSH consumption in poor responders, but conflicting data in relation to oocyte yield and implantation rates. The impact of letrozole co-treatment on hormonal changes and reproductive outcome after co-treatment in normal responders remains to be clarified. Study design, size, duration A multicentre double-blinded randomised placebo-controlled trial conducted in 4 fertility clinics at university hospitals in Denmark from August 2016 to November 2018. 159 women were randomised and 129 completed the study; 67 women in the letrozole group and 62 women in the placebo group. The study was conducted in accordance with the Helsinki Declaration and the ICH-Good-Clinical-Practice. Data collection and reporting followed the guidelines of CONSORT to achieve transparent reporting of trials. Participants/materials, setting, methods Women with expected normal ovarian reserve received an antagonist IVF protocol with fixed-dose FSH and fresh single embryo transfer. Co-treatment consisted of once-daily 5 mg letrozole or placebo from the start of stimulation until the day of triggering final oocyte maturation with human chorionic gonadotropin. Serum was collected on 7 visits from stimulation start to 8 days after oocyte retrieval. Clinical pregnancy was determined with a viable foetus by vaginal ultrasound at gestational week 7. Main results and the role of chance The proportion of patients with progesterone >1.5 ng/ml in the late follicular phase was similar in the letrozole versus placebo group with 6% versus 0%, respectively (OR 0, 95 % CI [0;1.6], P =.12). Mid-luteal progesterone levels >30 ng/ml were observed in 59% versus 31%, respectively, of subjects in the letrozole and placebo group (OR 3.3, 95% CI [1.4;7.1], P =.005). Letrozole treatment decreased estradiol levels by 69% (95 % CI [60%;75%], P Limitations, reasons for caution The diurnal variation of progesterone has been confirmed since this study was completed, hence the timing of the blood samples was not standardized . However, bias is unlikely due to the randomized design. The study was not powered to show an effect on ongoing pregnancy rates. Wider implications of the findings Letrozole co-treatment during ovarian stimulation with gonadotropins maintained serum estradiol at physiological levels, increased follicular phase levels of gonadotropins and androgens, and luteal progesterone levels. These data indicate that letrozole co-treatment may ameliorate the detrimental impacts of gonadotropin stimulation during IVF in normal responders. Trial registration number NCT02939898 and NCT02946684

Study question Can cooling of embryos during the embryo transfer be alleviated with the use of a 37 °C temperature protective device covering the ET catheter? Summary answer Cooling of embryos during embryo transfer can be effectively alleviated by using a 37 °C pre-warmed temperature protective device covering the ET catheter. What is known already An optimized physicochemical environment is crucial for maintenance of normal homeostasis, metabolism, and spindle stability to minimize stress on gametes and embryos. During preimplantation embryo development, epigenetic reprogramming occurs and environmental stress factors including temperature can disrupt this critical process and potentially damage embryos. IVF laboratories use heated stages, warming blocks and incubators to control and maintain temperature within set control limits. However, it has recently been shown that during the ET procedure, the temperature of fluid in the catheter tip drops significantly. To date no means of preventing this has been reported, or to our knowledge, implemented. Study design, size, duration In this prospective controlled study, 100 simulated embryo transfer procedures were carried out at 5 European clinics. The catheters were loaded with medium according to clinic protocol. In 50, the transfer catheter was then transported to the clinician and handled according to standard practice, and in the other 50 the catheter was covered with the temperature protecting device after loading but otherwise handled identically. 10 control and 10 intervention procedures were performed at each clinic. Participants/materials, setting, methods The temperature inside the ET catheter tip (Wallace Sure View) was measured with a calibrated thermocouple probe (diameter of 0.25 mm) placed at the location of an embryo and monitored during standard operating ET procedures (control group), and with the ET catheter-syringe set inserted into a temperature protective device (37 °C pre-warmed aluminium core, 15x90 mm) allowing retraction of the ET catheter tip immediately after embryo loading (study group). No embryos were employed in this study. Main results and the role of chance During standard operating ET procedures (control group), a considerable variation was observed in the embryo loading temperature between clinics, ranging from 34 °C to 37 °C. A profound temperature drop down to 20.8 °C–25.6 °C was recorded within 20 seconds of loading the ET catheter and in all 5 clinics a very rapid decline in catheter tip temperature down to ambient temperature was observed regardless of environment, type of workstation, or standard operating ET procedures in use. In contrast, when the ET catheter-syringe set was placed into a 37 °C pre-warmed temperature protective device from the time of embryo loading until the end of the simulated ET procedure, the drop of temperature was minimal, effectively maintaining the temperature at the loading temperature of between 34 °C and 37 °C °C throughout the simulated ET procedure. The mean loss of temperature of 14.8 °C in the control group was reduced to just to 0.4 °C in the study group. The consistent and profound differences in catheter tip temperature between the control and device groups across repeated measurements at different sites indicate the findings to be robust. Limitations, reasons for caution Numerous permutations of laboratory culture systems exist and the equipment, consumables and procedure for ET, including time, are highly variable and operator dependent. Therefore, the results and conclusions of this study may not be universally applicable. Furthermore, the impact of embryo cooling during ET on live birth rate remains uncertain. Wider implications of the findings: The ET procedure represents a ‘weak link’ in temperature control from the IVF laboratory to the patient until the embryo is safely deposited into its physiological environment, the uterine cavity. We demonstrate the effectiveness of a novel device for maintaining the temperature during ET, which could potentially improve embryo viability. Trial registration number Not applicable

Study question Does adjuvant letrozole in ovarian stimulation (OS) for in vitro fertilization (IVF) decrease the uterine peristalsis frequency (UPF) prior to fresh embryo transfer (ET)? Summary answer Adjuvant letrozole in (OS) for IVF does not reduce the UPF significantly prior to fresh ET. What is known already Throughout the cycle UPF aids spermatozoa transport to the fallopian tube and may affect implantation. At fresh, ET UPF is negatively correlated with implantation- and clinical pregnancy rates and is believed to be modulated by estradiol and progesterone. High levels of estradiol, from multiple follicular development, in OS have been reported to increase UPF, whereas progesterone is considered to be utero-relaxant. The influence of androgens is unclear. Co-treatment with letrozole during gonadotropin OS limits the estradiol rise the supra-physiological estradiol and may therefore reduce UPF prior to fresh ET. Study design, size, duration: This single centre study was nested within a multicentre double blinded RCT investigating the impact of letrozole co-treatment during gonadotropin OS for IVF on late follicular and luteal estradiol, progesterone and testosterone levels. Between 2016 and 2017, 39 women expected normal responders were randomised to co-treatment with letrozole or placebo. Of these, 33 women completed this element of the study. The study was carried out according to the Helsinki Declaration and the ICH-Good-Clinical-Practice. Participants/materials, setting, methods Eligible women were randomised 1:1 to adjuvant treatment with letrozole 5 mg/day or placebo in an antagonist protocol using a fixed dose of recFSH 150 IU/day. Final maturation was triggered with rhCG 6,500 IU and luteal support with vaginal progesterone was administered from the day following oocyte aspiration. Less than one hour prior to fresh ET, six minute duration transvaginal ultrasound recordings of the uterus in sagittal section were performed and blood samples were drawn. Main results and the role of chance A total of 33 women completed the study (letrozole n = 17; placebo n = 16). Age, BMI, and ovarian reserve markers were similar between the groups. On day of ET, serum estradiol levels were significantly suppressed in the letrozole group to mean 867 ± 827 pmol/L compared to 3,110 ± 1,528 pmol/L in the placebo group (P Limitations, reasons for caution The limited sample size risks masking minor effects. Wider implications of the findings: The supra-physiological levels of estradiol were significantly supressed in the intervention group, but UPF prior to fresh ET was similar in both groups. UPF is not strongly correlated to luteal phase sex steroid levels. Any beneficial effect of adjuvant letrozole during OS is not through an impact of UPF. Trial registration number NCT02939898

Study question The aim of this study was to evaluate the influence of blastocoele re-expansion time of warmed vitrified blastocysts on clinical pregnancy outcome. Summary answer Clinical pregnancy rate was significantly higher after transfer of warmed vitrified blastocysts that were fully expanded within 2 hours post thaw. What is known already The number of blastocysts being vitrified worldwide has increased dramatically over recent years. A combination of factors has led to this including the introduction of vitrification, an increase in freeze-all policies, single embryo transfer and an increase in preimplantation genetic testing. Currently, blastocyst re-expansion after thawing is used to indicate the survival status of the blastocyst and when combined with the morphology of blastocyst can predict its reproductive potential. While time taken for blastocoele re-expansion has been proposed to be a biomarker of viability, its value in clinical practice remains unclear. Study design, size, duration This retrospective study analysed outcomes in patients who had frozen embryo transfers between June-December 2020. 233 embryos were reviewed with time-lapse to assess their blastocoele expansion post-warming and three groups were identified. The first included fully expanded blastocysts post-warming. The second group included partially expanded blastocysts and the third non-expanded blastocysts. In addition, the groups were subcategorised into two further categories depending on whether they took less or more than 2 hours to complete expansion. Participants/materials, setting, methods 233 vitrified/warmed embryos from 216 patients were analysed using time-lapse incubators. The first group included 134 blastocysts, of which 70 were fully expanded within 2 hours and 64 after 2 hours post thaw. The second group had 70 embryos of which 45 expanded partially within 2 hours and 25 after 2 hours. The third had 28 embryos that had no expansion within the first 2 hours (n = 20) or after 2 hours (n = 8). Main results and the role of chance Blastocysts were collapsed by laser prior to vitrification. Single blastocyst transfer was performed for all patients. The mean transferred embryo age was 32.1± 5.5 and the recipient’s was 37.5± 5.9. Fully expanded blastocysts (n = 70) within 2 hours demonstrated a clinical pregnancy rate (CPR) of 57% compared with 38% from those that expanded fully after 2 hours (n = 64) (p = 0.02). Blastocysts with some form of expansion (full or partial) within 2 hours post-warming (n = 115) were associated a significantly higher CPR compared to those expanding after 2 hours (n = 89). The CPR was 55% and 39% respectively (p = 0.02). Embryos that showed no expansion (n = 20) within the first 2 hours post thaw resulted in CPR of 28%. Interestingly, embryos that showed no expansion after 2 hours resulted in no pregnancy. When combining morphology as a selection criterion, expansion within 2 hours of thawing was associated with a CPR of 62.5% for ≥4AB embryos, 50% for BB embryos and 45% for poorer embryos ≤CB.In conclusion, failure of blastocoele expansion post 2 hours reduced by half the chances of clinical pregnancy (p = 0.03). Combination of the degree of re-expansion and embryo morphology is an important predictor tool to improve clinical outcomes in frozen embryo transfers. Limitations, reasons for caution This study uses a small sample size of patients. The data are observational and were retrospectively analysed so unknown confounders could not be assessed. The addition of more cycles and further multivariate analysis, is essential for confirmation of the findings. However, initial results are very reassuring. Wider implications of the findings: The degree of speed of re-expansion post warming should be used as a predictor for prioritisation of embryos for transfer. Owing to these preliminary findings there is rationale for a larger scale study combining other morphological indicators that could further assess implantation indicators and assist patient counselling Trial registration number Not applicable

STUDY QUESTION What is the cumulative incidence of live birth and mean time to pregnancy (by conception after IVF/ICSI or natural conception) in women experiencing unexplained recurrent implantation failure (RIF) following IVF/ICSI treatment? SUMMARY ANSWER In 118 women who had experienced RIF, the reported cumulative incidence of live birth during a maximum of 5.5 years follow-up period was 49%, with a calculated median time to pregnancy leading to live birth of 9 months after diagnosis of RIF. WHAT IS KNOWN ALREADY Current definitions of RIF include failure to achieve a pregnancy following IVF/ICSI and undergoing three or more fresh embryo transfer procedures of one or two high quality embryos or more than 10 embryos transferred in fresh or frozen cycles. The causes and optimal management of this distressing condition remain uncertain and a range of empirical and often expensive adjuvant therapies is often advocated. Little information is available regarding the long-term prognosis for achieving a pregnancy. STUDY DESIGN, SIZE, DURATION Two hundred and twenty-three women under 39 years of age who had experienced RIF without a known cause after IVF/ICSI treatment in two tertiary referral university hospitals between January 2008 and December 2012 were invited to participate in this retrospective cohort follow up study. PARTICIPANTS/MATERIALS, SETTING, METHODS All eligible women were sent a letter requesting their consent to the anonymous use of their medical file data and were asked to complete a questionnaire enquiring about treatments and pregnancies subsequent to experiencing RIF. Medical files and questionnaires were examined and results were analysed to determine the subsequent cumulative incidence of live birth and time to pregnancy within a maximum 5.5 year follow-up period using Kaplan Meier analysis. Clinical predictors for achieving a live birth were investigated using a Cox hazard model. MAIN RESULTS AND THE ROLE OF CHANCE One hundred and twenty-seven women responded (57%) and data from 118 women (53%) were available for analysis. During the maximum 5.5 year follow up period the overall cumulative incidence of live birth was 49% (95% CI 39–59%). Among women who gave birth, the calculated median time to pregnancy was 9 months after experiencing RIF, where 18% arose from natural conceptions. LIMITATIONS, REASONS FOR CAUTION Since only 57% of the eligible study cohort completed the questionnaire, the risk of response bias limits the applicability of the study findings. WIDER IMPLICATIONS OF THE FINDINGS This study reports a favorable overall prognosis for achieving live birth in women who have previously experienced RIF, especially in those who continue with further IVF/ICSI treatments. However since 51% did not achieve a live birth during the follow-up period, there is a need to distinguish those most likely to benefit from further treatment. In this study, no clinical factors were found to be predictive of those achieving a subsequent live birth. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the University Medical Center Utrecht, in Utrecht and the Academic Medical Centre, in Amsterdam. NSM has received consultancy and speaking fees and research funding from Ferring, MSD, Merck Serono, Abbott, IBSA, Gedion Richter, and Clearblue. During the most recent 5-year period BCJMF has received fees or grant support from the following organizations (in alphabetic order); Actavis/Watson/Uteron, Controversies in Obstetrics & Gynecology (COGI), Dutch Heart Foundation, Dutch Medical Research Counsel (ZonMW), Euroscreen/Ogeda, Ferring, London Womens Clinic (LWC), Merck Serono, Myovant, Netherland Genomic Initiative (NGI), OvaScience, Pantharei Bioscience, PregLem/Gedeon Richter/Finox, Reproductive Biomedicine Online (RBMO), Roche, Teva, World Health Organisation (WHO). None of the authors have disclosures to make in relation to this manuscript.

STUDY QUESTION What is the effect of the positive reappraisal coping intervention (PRCI) on anxiety in women awaiting the outcome of an IVF/ICSI cycle? SUMMARY ANSWER Women reported significantly more anxiety during the waiting period than before treatment, but the use of the PRCI did not significantly reduce anxiety during the waiting period. WHAT IS KNOWN ALREADY Waiting for the outcome of IVF/ICSI treatment after embryo transfer is one of the most stressful periods of fertility treatments. At present, no evidence-based coping interventions are available to assist women though this waiting period. The PRCI has been designed to address this unmet need by promoting positive reappraisal coping. STUDY DESIGN, SIZE, DURATION A three-armed randomized controlled trail (RCT) was designed to evaluate the PRCI in women undergoing IVF/ICSI. Data were collected between October 2010 and June 2012. A total of 377 participants were randomized to receive either the PRCI and emotional monitoring, emotional monitoring only, or routine care. Only the PRCI-monitoring group received the coping intervention, comprising an explanatory leaflet and ten statements to be read at least once in the morning and once in the evening. PARTICIPANT, MATERIALS, SETTING, METHODS To capture the general impact of the PRCI, all three groups completed questionnaires at three time points: just before the waiting period (time 1: stimulation phase), on Day 10 of the 14-day waiting period (time 2: waiting period) and 6 weeks after the start of the waiting period (time 3: 6-week follow-up). In addition, to capture the specific impacts of the PRCI on the days of the waiting period, the PRCI-monitoring group and the monitoring-control group also rated their emotions and reactions daily, for the 14-day waiting period. MAIN RESULTS AND THE ROLE OF CHANCE Of the women who agreed to participate and who met eligibility criteria, 377 were randomized. All study participants reported significantly more anxiety and depression during the waiting period than before treatment (P < 0.001). The mean difference in anxiety between time 1 versus time 2 was 1.465 (95% CI 1.098–1.832). The mean difference in depression between time 1 versus time 2 was 0.514 (95% CI 0.215–0.813). Use of the PRCI did not significantly reduce anxiety or depression, or daily negative emotions during the waiting period. However, patients randomized to the PRCI reported significantly more positive emotions during the waiting period (P < 0.001) than the monitoring-control group, and reported the intervention to be easy to use, and as having a positive psychological effect. No significant differences were found between the groups in treatment outcome. LIMITATIONS, REASONS FOR CAUTION The lack of difference observed in the present study for anxiety levels between the PRCI and the monitoring-control group could have been due to the effects of monitoring itself or its ability to attenuate or obscure the effects of the PRCI intervention in unknown ways. A randomized group of women who used only the PRCI without daily monitoring would provide more insight. WIDER IMPLICATIONS OF THE FINDINGS The PRCI was shown to help women reinterpret the demands of the waiting period in a more positive way. These results are consistent with previous studies showing that positive reappraisal coping is a useful strategy for unpredictable and uncontrollable situations represented by a medical waiting period. This simple low cost self-help coping intervention increases positive affect during the waiting period in an IVF/ICSI treatment.

study question: How do the expression patterns of neuronal markers differ in the endometrium of women with and without endometriosis? summary answer: The neuronal markers, PGP9.5, NGFp75 and VR1, are expressed in the endometrium at levels that do not differ between women with and without endometriosis. what is known already: Aberrant neuronal growth within the uterus may contribute to abnormal fertility and uterine dysfunction. However, controversy still exists as to whether aberrant innervation in the endometrium is associated with gynaecological pathology such as endometriosis. This may reflect the use of subjective methods such as histology to assess the innervation of the endometrium. We, therefore, employed a quantitative method, western blotting, to study markers of endometrial innervation in the presence and absence of endometriosis. study design, size, duration: This study included 45 women undergoing laparoscopic examination for the diagnosis of endometriosis. Endometrial samples were analysed by western blot for the expression of neuronal and neurotrophic markers, PGP9.5, VR1 and NGFp75. participants/materials,settings,methods: Endometrial pipelle biopsies were obtained from patients with (n ¼ 20, study group) and without (n ¼ 25, control group) endometriosis. Tissue was analysed by immunohistochemistry and western blot analysis for the expression of pan-neuronal marker, PGP9.5, sensory nociceptive marker, TPVR1, and low-affinity neurotrophic growth factor receptor, NGFRp75. mainresultsandtheroleofchance: PGP9.5, NGFp75 and VR1 were expressed in the endometrium of women, independent of the presence of endometriosis. Furthermore, the expression level of PGP9.5, VR1 and NGFp75 did not alter between the two cohorts of women. limitations,reasonsforcaution: Studies of this nature are subject to the heterogeneous nature of patient population and tissue samples despite attempts to standardize these parameters. Hence, further studies using similar methodology will be required to confirm our results. widerimplicationsofthefindings: Our results highlight that sensory neuronal markers are present in women with and without endometriosis. Future work will assess what the targets of the endometrial nerves are and investigate their function, their impact on endometrial biology and, in particular, whether aberrant neuronal function, rather than the mere presence of neuronal function, could be the root cause of subfertility and/or pain affecting many endometriosis sufferers. Our results do not, however, confirm the previous paradigm of increased innervation in the endometrium of women with endometriosis, nor the use of nerve cell detection from pipelle biopsies to diagnose endometriosis. study funding/competing interest(s): This work was funded by the Infertility Research Trust (IRT) and the University of Malaya. There are no competing interests.

Are daily cycles in urinary melatonin and oxidative stress marker levels (8-hydroxydeoxyguanosine) altered in PCOS, and is this associated with changes in sleep quality?There is an association between elevated nighttime melatonin and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, and poor sleep quality in our PCOS study group.Women with PCOS are known to have poorer sleep. However, there have been few studies examining the possible association between melatonin levels and sleep quality in women with polycystic ovarian syndrome (PCOS).This is a case-control study of PCOS (n = 26) and non-PCOS control (n = 26) subjects recruited from a tertiary gynaecological centre.The participants were requested to complete sleep questionnaires for a month. In a subgroup from these cohorts (PCOS, n = 15; controls, n = 18), urine samples were also collected at various time points over a 24-h period. In addition, their sleep patterns and lighting environment were monitored for 3 consecutive days and nights using a wrist-mounted Actiwatch device.PCOS women had significantly elevated night-time urinary levels of the melatonin metabolite 6-sulfatoxymelatonin (aMT6s) and of 8-OHdG (both at P0.05), as well as significantly reduced sleep quality (P0.05), compared with the controls.Due to the small sample size of the study, further studies will be required to confirm our findings.Our preliminary work provides a possible new insight into the interactions between melatonin, increased oxidative stress and sleep in women with PCOS.The study was funded by the Faculty of Medicine, University of Southampton.

Polycystic ovary syndrome (PCOS) is a complex disorder with variable prevalence and clinical presentation in different populations, which may be mediated by geographical and ethnic background.We performed a comparison of phenotypic characteristics between 547 Chinese and 427 Dutch women with PCOS and oligo/amenorrhoea attending University Reproductive Centers in China and the Netherlands.Chinese women presenting with a clinical diagnosis of PCOS were observed to have a higher incidence of hyperandrogenism (HA) (P0.001) and amenorrhoea (P0.001) compared with Dutch women, but no difference was observed in the incidence of polycystic ovaries (PCOs). Using population-specific cut-off values, Chinese women with PCOS demonstrated a higher incidence of increased BMI (P0.001), waist circumference (WC) (P0.001) and waist-hip ratio (P0.001) than Dutch women. In both groups, HA was associated with increased age, fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR) and serum LH while PCOs correlated with BMI, WC, HOMA-IR, fasting insulin and elevated total testosterone. Associations specific for ethnic background were found between LH and HA, and between both BMI and HOMA-IR, and PCOs.Reproductive and metabolic characteristics differed between the two ethnic groups. Chinese women were found to present more frequently with a phenotype associated with increased risk of metabolic complications later in life, compared with Dutch Caucasian women. Ethnicity seems to determine part of the specific phenotypical presentation of PCOS.

Background: the use of housekeeping genes (HKG) as internal controls for real-time qPCR studies of gene expression is based on the assumption of their inherent stability. However, it is unclear whether this stability is maintained in disease states. In order to test this, the present study investigated the expression of specific HKG in the endometrium of healthy and polycystic ovarian syndrome(PCOS) women. Methods: endometrial tissue samples were taken from women with PCOS (n= 9) and controls (n= 10). The stability of nine candidate reference genes in the endometrial tissues were evaluated; four encode mitochondrial proteins [ATP5B, succinate dehydrogenase complex subunit A (SDHA), cytochrome c-1, glyceraldehyde-3-phosphatedehydrogenase], two encode ribosomal protein genes (18s ribosomal RNA, ribosomal protein L13A), one for cell structure (SDHA), one for cell signalling (beta actin, ACTB) and one involved in DNA repair (topoisomerase I, TOP1). The expression stability of these HKGs was calculated using geNORM qbasePLUS software, with stability defined by M-values, where higher M-value indicating less stability. In addition, changes in their cycle threshold values were analysed to determine direction of change between groups, and a Mann-Whitney U-test was used to determine statistical differences in expression. Results: the most stable HKGs observed across both PCOS endometrium were found to be YWHAZ, CYC1 and ACTB. Further TOP1 demonstrated higher gene expression in the endometrium from PCOS women compared with those from healthy women. Conclusions: of the nine HKGs examined, only YWHAZ, CYC1 and ACTB were stable in both control and PCOS endometrium: these should therefore be used as internal controls for quantitative reverse transcription-polymerase chain reaction analysis. Published discrepancies between endometrial gene expression studies may therefore be due in part to in the inappropriate HKG selection, and future gene expression studies should be based on HKG of known stability in both the disease and healthy states to avoid erroneous interpretation of results

BACKGROUND A proportion of women with ‘unexplained’ infertility may present with subfertility because their pregnancies fail before they are clinically recognized. In order to test whether pre-clinical early pregnancy losses (EPL) occur more frequently in women with unexplained infertility, serial urinary hCG concentrations were measured to compare EPL per cycle rates following spontaneous conception in patients with unexplained infertility versus healthy volunteers. METHODS Sixty patients under 39 years of age with unexplained infertility and 60 healthy controls, who were trying to conceive spontaneously, participated in this study. All participants were asked to collect daily urine samples from cycle day 14 until menstruation for three consecutive cycles or until a positive pregnancy test was obtained. Urinary hCG and creatinine levels were measured by immunoassay. Implantation was detected when urinary hCG levels rose above reference levels constructed from samples obtained from 12 women not attempting to conceive. EPL rates were determined by a linear mixed model using logarithmically transformed hCG/creatinine data. RESULTS In the 133 cycles of 60 women with unexplained infertility, just one implantation was detected, which became an ongoing pregnancy. In contrast, in 103 such cycles in 46 control patients, 30 implantations were detected (24 clinical pregnancies, 6 cases of EPL). The odds ratio for EPL/cycle in the unexplained versus control group was 0 (95% confidence interval: 0–0.795, P= 0.026). CONCLUSIONS Our data do not support the hypothesis that recurrent EPL may present as unexplained infertility. Post-implantation failure is therefore unlikely to contribute significantly to the presentation of subfertility.

BACKGROUND This study investigates whether dietary patterns, substantiated by biomarkers, are associated with semen quality. METHODS In 161 men of subfertile couples undergoing in vitro fertilization treatment in a tertiary referral clinic in Rotterdam, the Netherlands, we assessed nutrient intakes and performed principal component factor analysis to identify dietary patterns. Total homocysteine (tHcy), folate, vitamin B12 and B6 were measured in blood and seminal plasma. Semen quality was assessed by sperm volume, concentration, motility, morphology and DNA fragmentation index (DFI). Linear regression models analyzed associations between dietary patterns, biomarkers and sperm parameters, adjusted for age, body mass index (BMI), smoking, vitamins and varicocele. RESULTS The 'Health Conscious' dietary pattern shows high intakes of fruits, vegetables, fish and whole grains. The 'Traditional Dutch' dietary pattern is characterized by high intakes of meat, potatoes and whole grains and low intakes of beverages and sweets. The 'Health Conscious' diet was inversely correlated with tHcy in blood (beta = -0.07, P = 0.02) and seminal plasma (beta = -1.34, P = 0.02) and positively with vitamin B6 in blood (beta = 0.217, P = 0.01). An inverse association was demonstrated between the 'Health Conscious' diet and DFI (beta = -2.81, P = 0.05). The 'Traditional Dutch' diet was positively correlated with red blood cell folate (beta = 0.06, P = 0.04) and sperm concentration (beta = 13.25, P = 0.01). CONCLUSIONS The 'Health Conscious' and 'Traditional Dutch' dietary pattern seem to be associated with semen quality in men of subfertile couples.

The study of human endometrial-embryonic interactions is complicated by the disruptive impact of endometrial sample collection on the process of implantation itself. Endometrial secretion analysis is a novel technique, non-disruptive to implantation. The primary aim of this prospective cohort study was to explore whether a cytokine profile predictive of implantation and clinical pregnancy can be identified in endometrial secretions aspirated immediately prior to embryo transfer following IVF.Endometrial secretions, aspirated immediately prior to embryo transfer from 210 women undergoing IVF, were analyzed using a multiplex immunoassay for 17 soluble regulators of implantation, namely interleukin (IL)-1beta, IL-5, IL-6, IL-10, IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, macrophage migration inhibitory factor, eotaxin, IFN-gamma-inducible 10 kDa protein (IP-10), monocyte chemo-attractant protein-1 (MCP-1), Dickkopf homolog 1, heparin-binding epidermal growth factor and vascular endothelial growth factor (VEGF). In order to detect implantation, daily urine samples were collected after embryo transfer, and human Chorionic Gonadotropin (hCG) concentrations were analyzed by an immunoassay.Multivariable logistic regression analysis revealed significant associations (negative and positive association, respectively) between MCP-1 (P = 0.005) and IP-10 (P = 0.037) levels and implantation, and between IL-1beta (P = 0.047) and TNF-alpha (P = 0.023) levels and clinical pregnancy. The predictive value for pregnancy of IL-1beta and TNF-alpha was observed to be equivalent and additive to that of embryo quality.Endometrial secretion cytokine profiling offers a novel, non-disruptive approach to study the role of the endometrium in human embryo implantation and identifies a profile which appears to be conducive to clinical pregnancy.www.clinicaltrials.gov (nCT00264992).

Background: Maternal hyperhomocysteinemia is detrimental for reproduction, but the effects on embryo quality are unknown. The aim of this study was to investigate whether biomarkers of the homocysteine pathway are associated with in vitro fertilization (IVF) outcome. METHODS: In a prospective study, we investigated biomarkers of the homocysteine pathway for associations with embryo quality and biochemical pregnancy in women undergoing IVF or intracytoplasmic sperm injection treatment (n = 181). In the treatment cycle, blood and monofollicular fluid samples were collected for determination of folate, cobalamin and total homocysteine (tHcy) concentrations. Results: Of all the women in the study, 67% used folic acid supplements. In blood, a significant correlation was established between high cobalamin and better embryo quality [standardized adjusted regression coefficient: -0.17, 95% confidence interval (CI): -0.30, -0.01]. In monofollicular fluid of non-supplemented women, high cobalamin correlated with better embryo quality (estimate: -0.87; 95% CI: -1.68, -0.06), whereas high tHcy resulted in poor embryo quality (estimate: 1.01; 95% CI: 0.08, 1.95). However, in monofollicular fluid of supplemented women, high tHcy correlated with better embryo quality (estimate: -0.58; 95% CI: -1.12, -0.04). In the total group, a 2-fold increase of monofollicular fluid folate corresponded with a 3.3 times higher chance (95% CI: 1.09, 9.71) of achieving pregnancy. Conclusions: An optimal homocysteine pathway in follicular fluid is associated with a better embryo quality and chance of pregnancy.

BACKGROUND: Ovarian stimulation gives rise to supraphysiological estradiol levels, which may affect oocyte quality. This study aims to investigate whether ovarian stimulation deranges the homocysteine pathway thereby affecting the pre-ovulatory follicle. METHODS: Blood samples were collected on cycle day 2 and the day of hCG administration in 181 women undergoing ovarian stimulation for IVF. In each subject, the diameter of the two leading follicles was measured and the corresponding follicular fluids were collected. In blood and follicular fluid samples, total homocysteine (tHcy), folate, cobalamin and pyridoxal'5-phosphate (PLP) were determined. According to the blood folate levels, women were classified as either folic acid supplemented (n = 113) or non-supplemented (n = 32). RESULTS: Ovarian stimulation resulted in a significant decrease in blood tHcy and cobalamin levels (both P

BACKGROUND: Cumulative IVF pregnancy rates are compromised by the large number of couples who drop-out of treatment before achieving pregnancy. The aim of this study was to identify the role of the treatment strategy applied, and potential other factors that influence the decision of couples to discontinue treatment. METHODS: The incidence of drop-out from WIT treatment and factors related to drop-out were studied in a cohort of IVF patients aged 50%. The presence of severe male subfertility (HR 4.80; 95% CI, 1.63-14.13) and the failure to achieve embryo transfer (odds ratio 0.41; 95% CI, 0.24-0.72) were also related to drop-out. CONCLUSIONS: Reducing drop-out rate is crucial to further improve the efficacy and cost-effectiveness of IVF treatment. An important factor determining the risk of drop-out is the burden of the treatment strategy. The application of a mild treatment strategy and managing patient's expectations might reduce drop-out rates.

BACKGROUND Conventional ovarian stimulation and the transfer of two embryos in IVF exhibits an inherent high probability of multiple pregnancies, resulting in high costs. We evaluated the cost-effectiveness of a mild compared with a conventional strategy for IVF. METHODS Four hundred and four patients were randomly assigned to undergo either mild ovarian stimulation/GnRH antagonist co-treatment combined with single embryo transfer, or standard stimulation/GnRH agonist long protocol and the transfer of two embryos. The main outcome measures are total costs of treatment within a 12 months period after randomization, and the relationship between total costs and proportion of cumulative pregnancies resulting in term live birth within 1 year of randomization. RESULTS Despite a significantly increased average number of IVF cycles (2.3 versus 1.7; P < 0.001), lower average total costs over a 12-month period (8333 versus euro10 745; P = 0.006) were observed using the mild strategy. This was mainly due to higher costs of the obstetric and post-natal period for the standard strategy, related to multiple pregnancies. The costs per pregnancy leading to term live birth were euro19 156 in the mild strategy and euro24 038 in the standard. The incremental cost-effectiveness ratio of the standard strategy compared with the mild strategy was euro185 000 per extra pregnancy leading to term live birth. CONCLUSIONS Despite an increased mean number of IVF cycles within 1 year, from an economic perspective, the mild treatment strategy is more advantageous per term live birth. It is unlikely, over a wide range of society's willingness-to-pay, that the standard treatment strategy is cost-effective, compared with the mild strategy.

BACKGROUND: Psychological variables, such as anxiety and depression, may have a negative impact on IVF outcomes, but the evidence remains inconclusive. Previous studies have usually measured a single psychological parameter with clinical pregnancy as the outcome. The objective of the current study was to determine whether pretreatment or procedural psychological variables in women undergoing a first IVF cycle affect the chance of achieving a live birth from that cycle. METHODS: Between February 2002 and February 2004, 391 women with an indication for IVF were recruited at two University Medical Centres in The Netherlands. Pretreatment anxiety and depression were measured with the Hospital Anxiety and Depression Scale. The Daily Record Keeping Chart was used to measure negative and positive affect before treatment and daily during ovarian stimulation. Multiple stepwise forward logistic regression analysis was performed with term live birth as the dependent variable. RESULTS: Regression analysis showed that women who expressed less negative affect at baseline were less likely to achieve live birth (P = 0.03). After one IVF cycle, women who received a standard IVF strategy were more likely to reach live birth delivery than those who received a mild IVF strategy (P = 0.002). A male/female indication for IVF was associated with a higher chance of achieving term live birth than a female only indication (P = 0.03). Age, duration of infertility or type of infertility were not independent predictors of live birth. CONCLUSIONS: The relationship between psychological parameters and IVF success rates is more complex than commonly believed. The expression of negative emotions before starting IVF might not be always detrimental for outcomes.

BACKGROUND: Failure of IVF treatment after a number of cycles can be devastating for couples. Although mild IVF strategies reduce the psychological burden of treatment, failure may cause feelings of regret that a more aggressive approach, including the transfer of two embryos, was not employed. In this study, the impact of treatment failure after two or more cycles on stress was studied, following treatment with a mild versus a standard treatment strategy. METHODS: Randomized controlled two-centre trial (ISRCTN35766970). Women were randomized to undergo mild ovarian stimulation (including GnRH antagonist co-treatment) and single embryo transfer (n 5 197) or standard GnRH agonist long-protocol ovarian stimulation with double embryo transfer (n 5 194). Participants completed the Hospital Anxiety and Depression Scale prior to commencing treatment and 1 week after the outcome of their final treatment cycle was known. Data from women who underwent two or more IVF cycles were subject to analysis (n 5 253). RESULTS: Women who experienced treatment failure after standard IVF treatment presented more symptoms of depression 1 week after treatment termination compared with women who had undergone mild IVF: adjusted mean (+ +95% confidence interval)510.2 (+2.3) versus 5.4 (+1.8), respectively, P 5 0.01. CONCLUSIONS: Failure of IVF treatment after a mild treatment strategy may result in fewer short-term symptoms of depression as compared to failure after a standard treatment strategy. These findings may further encourage the application of mild IVF treatment strategies in clinical practice.

textabstractBackground: Milder stimulation protocols are being developed to minimize adverse effects of ovarian stimulation in in vitro fertilization (IVF) programs. A drawback is the possibility of an increased rate of insufficient ovarian response. This study aimed to develop a prognostic model for the prediction of cycle cancellation due to insufficient response to mild stimulation. Methods: A total of 174 IVF patients aged

BACKGROUND: In order to assess the frequency of aneuploidy and mosaicism in embryos obtained from IVF patients aged

BACKGROUND: The objective of this study was to assess the psychological implications of mild ovarian stimulation combined with single embryo transfer (SET) during a first IVF cycle. METHODS: We conducted a randomized controlled two-centre trial. Three hundred and ninety-one couples were randomized to undergo either mild ovarian stimulation with GnRH antagonist co-treatment and SET (n=199) or conventional GnRH agonist long protocol ovarian stimulation with double embryo transfer (DET) (n=192). Women completed the Hospital Anxiety and Depression Scale, the Hopkins Symptom Checklist and the Subjective Sleep Quality Scale at baseline, on the first day of ovarian stimulation and following embryo transfer. Affect was assessed daily with the Daily Record Keeping Chart from the first day of ovarian stimulation until the day treatment outcome became known. RESULTS: The conventional IVF group experienced elevated levels of physical and depressive symptoms during pituitary downregulation. At oocyte retrieval, this group experienced more positive affect and less negative affect than the mild IVF group. In the conventional IVF group, cycle cancellation was associated with less positive and more negative affect. CONCLUSIONS: During the first IVF treatment cycle, mild ovarian stimulation and SET does not lead to more psychological complaints than conventional IVF.

Item does not contain fulltext BACKGROUND: Specific subgroups of people planning IVF might be at risk of having more psychological or health-related problems. Identification of subgroups at risk may better enable allocation of appropriate counselling. METHODS: A group of 425 men and 447 women planning to undergo IVF treatment filled out a questionnaire. Four domains of health-related quality of life were measured, namely perceived emotional, physical, cognitive and social functioning. RESULTS: Young men and women (aged 21-30 years) planning IVF had more short-term social and emotional problems than people of the same age group in the general population. No substantial differences were found in cognitive and physical functioning for all age groups of men nor women planning IVF compared with the general population. A high level of irrational parenthood cognitions substantially accounted for a less optimal score on all the different domains of quality of life. These cognitions ('needing a child in order to live a happy life') were especially prevalent among younger women. CONCLUSIONS: Patients with high levels of irrational parenthood cognitions are at risk of a less optimal quality of life. A short cognitive counselling therapy is advised for patients with high levels of these cognitions.

BACKGROUND: Extending the period of in-vitro culture to the blastocyst stage may improve implantation rates in IVF treatment. Recognition of the dynamic nature of early embryo metabolism has led to the development of commercially available sequential culture systems. However, their improved efficacy over monoculture systems remains to be demonstrated in prospective studies. METHODS: Embryos obtained from 158 women undergoing IVF treatment were randomized by sealed envelopes to culture in one of three systems: (A) culture for 5 days in our own monoculture medium (Rotterdam medium); (B) culture for 3 days in Rotterdam medium followed by 2 days in fresh Rotterdam medium; (C) culture for 5 days using the commercially available G1/G2 sequential culture system. RESULTS: There were no significant differences in blastulation, implantation or pregnancy rates between the three tested culture systems. CONCLUSION: The employed monoculture system is as effective as the G1/G2 sequential system for the culture of blastocysts for IVF.

Changing the way in which successful IVF treatment is defined offers a tool to improve efficacy while reducing costs and complications of treatment. Crucial to this paradigm shift is the move away from considering outcomes in terms of the single IVF cycle, and towards the started IVF treatment as a whole. We propose the most informative end-point of success in IVF to be the term singleton birth rate per started IVF treatment (or per given time period) in the overall context of patient discomfort, complications and costs. These end-points are important not only for patients, but also for clinicians, health economists and policy makers. Such an approach would encourage the development of patient-friendly and cheaper stimulation protocols with less stress, discomfort and side effects. The combination of mild ovarian stimulation with single embryo transfer may provide the same overall pregnancy rate per total IVF treatment, achieved in the same amount of time for similar direct costs, but with reduced patient stress and discomfort, and the near complete elimination of multiple pregnancies. This would offer major health and indirect cost benefits. If IVF success rates were to be expressed in terms of delivery of a term single baby per IVF treatment (or in a given treatment period), the introduction of single embryo transfer on a large scale would be facilitated.