Idiopathic early ovarian ageing: risk of miscarriage and chance of delivery following ART in a nationwide cohort study.

STUDY QUESTION: Is idiopathic reduced ovarian reserve in young women, quantified as low response to ovarian stimulation in ART, associated with a concomitant loss of oocyte quality as determined by risk of pregnancy loss and chance of clinical pregnancy and live birth? SUMMARY ANSWER: Young women with idiopathic accelerated loss of follicles exhibit a similar risk of pregnancy loss as young women with normal ovarian reserve. WHAT IS KNOWN ALREADY: Normal ovarian ageing is described as a concomitant decline in oocyte quantity and quality with increasing age. Conflicting results exist with regard to whether a similar decline in oocyte quality also follows an accelerated loss of follicles in young women. STUDY DESIGN, SIZE, DURATION: This national register-based, historical cohort study included treatment cycles from young women (≤37 years) after ART treatment in Danish public or private fertility clinics during the period 1995-2014. The women were divided into two groups dependent on their ovarian reserve status: early ovarian ageing (EOA) group and normal ovarian ageing (NOA) group. There were 2734 eligible cycles in the EOA group and 22 573 in the NOA group. Of those, 1874 (n = 1213 women) and 19 526 (n = 8814 women) cycles with embryo transfer were included for analyses in the EOA and NOA group, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: EOA was defined as ≤5 oocytes harvested in both the first and second cycle stimulated with FSH. The NOA group should have had at least two FSH-stimulated cycles with ≥8 oocytes harvested in either the first or the second cycle. Cases with known causes influencing the ovarian reserve (endometriosis, ovarian surgery, polycystic ovary syndrome, chemotherapy, etc.) were excluded. The oocyte quality was evaluated by the primary outcome defined as the overall risk of pregnancy loss (gestational age (GA) ≤22 weeks) following a positive hCG and further stratified into: non-visualized pregnancy loss, early miscarriage (GA ≤ 12 weeks) and late miscarriage (GA > 12 weeks). Secondary outcomes were chance of clinical pregnancy and live birth per embryo transfer. Cox regression models were used to assess the risk of pregnancy loss. Time-to-event was measured from the day of embryo transfer from the second cycle and subsequent cycles. Logistic regression models were used to assess the chance of clinical pregnancy and live birth. MAIN RESULTS AND THE ROLE OF CHANCE: The overall risk of pregnancy loss for the EOA group was comparable with the NOA group (adjusted hazard ratio: 1.04, 95% CI: 0.86; 1.26). Stratifying by pregnancy loss types showed comparable risks in the EOA and NOA group. The odds of achieving a clinical pregnancy or live birth per embryo transfer was lower in the EOA group compared to the NOA group (adjusted odds ratio: 0.77 (0.67; 0.88) and 0.78 (0.67; 0.90), respectively). LIMITATIONS, REASONS FOR CAUTION: Only women with at least two ART cycles were included. We had no information on the total doses of gonadotropin administered in each cycle. WIDER IMPLICATIONS OF THE FINDINGS: The present findings may indicate that mechanism(s) other than aneuploidy may explain the asynchrony between the normal-for-age risk of miscarriage and the reduced chance of implantation found in our patients with EOA. The results of this study could be valuable when counselling young patients with low ovarian reserve. STUDY FUNDING/COMPETING INTERESTS(S): The study was funded by the Health Research Fund of Central Denmark Region. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Early ovarian ageing: is a low number of oocytes harvested in young women associated with an earlier and increased risk of age-related diseases?

STUDY QUESTION: Do young women with early ovarian ageing (EOA), defined as unexplained, and repeatedly few oocytes harvested in ART have an increased risk of age-related events? SUMMARY ANSWER: At follow-up, women with idiopathic EOA had an increased risk of age-related events compared to women with normal ovarian ageing (NOA). WHAT IS KNOWN ALREADY: Early and premature menopause is associated with an increased risk of cardiovascular diseases (CVDs), osteoporosis and death. In young women, repeated harvest of few oocytes in well-stimulated ART cycles is a likely predictor of advanced menopausal age and may thus serve as an early marker of accelerated general ageing. STUDY DESIGN, SIZE, DURATION: A register-based national, historical cohort study. Young women (≤37 years) having their first ART treatment in a public or private fertility clinic during the period 1995-2014 were divided into two groups depending on ovarian reserve status: EOA (n = 1222) and NOA (n = 16 385). Several national registers were applied to assess morbidity and mortality. PARTICIPANTS/MATERIALS, SETTING, METHODS: EOA was defined as ≤5 oocytes harvested in a minimum of two FSH-stimulated cycles and NOA as ≥8 oocytes in at least one cycle. Cases with known causes influencing the ovarian reserve (endometriosis, ovarian surgery, polycystic ovary syndrome, chemotherapy etc.) were excluded. To investigate for early signs of ageing, primary outcome was an overall risk of ageing-related events, defined as a diagnosis of either CVD, osteoporosis, type 2 diabetes, cancer, cataract, Alzheimer's or Parkinson's disease, by death of any-cause as well as a Charlson comorbidity index score of ≥1 or by registration of early retirement benefit. Cox regression models were used to assess the risk of these events. Exposure status was defined 1 year after the first ART cycle to assure reliable classification, and time-to-event was measured from that time point. MAIN RESULTS AND THE ROLE OF CHANCE: Median follow-up time from baseline to first event was 4.9 years (10/90 percentile 0.7/11.8) and 6.4 years (1.1/13.3) in the EOA and NOA group, respectively. Women with EOA had an increased risk of ageing-related events when compared to women with a normal oocyte yield (adjusted hazard ratio 1.24, 95% CI 1.08 to 1.43). Stratifying on categories, the EOA group had a significantly increased risk for CVD (1.44, 1.19 to 1.75) and osteoporosis (2.45, 1.59 to 3.90). Charlson comorbidity index (1.15, 0.93 to 1.41) and early retirement benefit (1.21, 0.80 to 1.83) was also increased, although not reaching statistical significance. LIMITATIONS, REASONS FOR CAUTION: Cycles never reaching oocyte aspiration were left out of account in the inclusion process and we may therefore have missed women with the most severe forms of EOA. We had no information on the total doses of gonadotrophin administered in each cycle. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that oocyte yield may serve as marker of later accelerated ageing when, unexpectedly, repeatedly few oocytes are harvested in young women. Counselling on life-style factors as a prophylactic effort against cardiovascular and other age-related diseases may be essential for this group of women. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this study. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Low-to-moderate alcohol consumption and success in fertility treatment: a Danish cohort study.

STUDY QUESTION: Does female weekly alcohol intake and binge drinking impact the chance of a successful fertility treatment? SUMMARY ANSWER: Low-to-moderate weekly alcohol drinking and binge drinking were not associated with the chance of achieving a clinical pregnancy or a live birth among women and couples undergoing medically assisted reproduction (MAR) treatments. WHAT IS KNOWN ALREADY: Alcohol consumption is common among women of reproductive age, even though health authorities advise women trying to conceive to abstain from drinking. A growing number of couples struggle with infertility, but it is unknown whether low-to-moderate levels of alcohol consumption and alcohol binge drinking impair success in fertility treatment. STUDY DESIGN, SIZE, DURATION: Cohort study with prospectively collected exposure information including 1708 women and potential partners undergoing fertility treatment at the public fertility clinic, Aarhus University Hospital, 1 January 2010 to 31 August 2015. In total, data on 1511 intrauterine insemination (IUI) cycles, 2870 in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles and 1355 frozen embryo transfer cycles. PARTTICIPANTS/MATERIALS, SETTING, METHODS: Exposure to weekly average alcohol intake was assessed from questionnaires completed by participants before the start of treatment. Outcome measures are the achievement of a clinical pregnancy and live birth in consecutive treatment cycles in the Danish national health registries, enabling complete follow-up. A modified Poisson regression with robust standard errors was used to evaluate associations between a weekly average alcohol intake and MAR outcomes, adjusting for female age, body mass index, cigarette smoking, coffee consumption, chronic diseases, level of education, and cycle number. When evaluating the association between binge drinking in the month prior to baseline and MAR outcomes the analyses were further adjusted for average weekly alcohol consumption. MAIN RESULTS AND THE ROLE OF CHANCE: Low-to-moderate average weekly alcohol intake was not statistically significantly associated with the chance of achieving a clinical pregnancy or a live birth following IUI or IVF/ICSI treatment cycles. Compared to women abstaining from alcohol, the adjusted relative risks for achieving a live birth among those reporting 1-2, 3-7, and >7 drinks per week were 1.00 (95% CI 0.66; 1.53), 1.20 (0.76; 1.91), and 1.48 (0.56; 3.93), respectively, among women initiating IUI treatments. Among those initiating IVF/ICSI treatments, the chance for achieving a live birth among those reporting 1-2, 3-7, and >7 drinks per week were 1.00 (0.83; 1.21), 0.95 (0.75; 1.20), and 0.89 (0.53; 1.51), respectively. The chance of achieving a live birth in the first IUI or IVF/ICSI treatment cycle was unrelated to the number of binge drinking episodes in the month preceding baseline. LIMITATIONS, REASONS FOR CAUTION: The risk of non-differential exposure misclassification, confounding, or chance cannot be ruled out. In addition, due to the low number of women reporting an intake of >7 drinks/week, the potential effect of high alcohol consumption should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: Although it remains unsettled if and how alcohol affects female reproduction, our results indicate that is not necessary to abstain from alcohol when striving for a successful outcome following fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): J.L. is supported by a fully financed Ph.D. scholarship from Aarhus University and has received funds from the A.P. Møller foundation. The funding sources had no involvement in the conduct of the article. Dr Kesmodel reports personal fees from MSD and Ferring Pharmaceuticals outside the submitted work. All other authors have no conflicts of interest to declare and all have completed the ICMJE disclosure form. TRIAL REGISTRATION NUMBER: Not relevant.

Elective embryo transfers on Day 6 reduce implantation compared with transfers on Day 5.

STUDY QUESTION: Is there a difference in pregnancy rates between embryos transferred electively on Day 5 and Day 6, respectively? SUMMARY ANSWER: The chance of pregnancy is significantly reduced (odds ratio (OR): 0.34; 95% CI 0.22-0.52) if transfer is performed on Day 6 compared with Day 5. WHAT IS KNOWN ALREADY: Several studies report that Day 5 transfers have higher implantation rates (IRs) when compared with Day 6 transfers. These studies were based on non-elective Day 6 transfers, where transfers on Day 6 were performed with developmentally delayed embryos. Traditionally, difference in IRs has therefore been explained by an impaired embryo quality. An alternative explanation is that endometrial receptivity is higher on Day 5 compared with Day 6. STUDY DESIGN, SIZE, DURATION: The study was conducted as a retrospective cohort follow-up study on single blastocyst transfers from February 2011 until August 2015 in patients aged <38 years, with ≥eight oocytes retrieved and no diagnosis of endometriosis. Non-elective Day 6 transfers were excluded. Post hoc power-calculations (two-sided level of significance 0.05, power of 0.80) indicate that 91 embryos were needed in each group to detect a reduction in IR (primary outcome) from 40 to 20%. PARTICIPANTS/MATERIALS, SETTING, METHODS: Day 5 or Day 6 transfers were implemented accordingly: from 2011 till 2013, transfers were performed on Day 6. If Day 6 was a Sunday, patients received Day 5 transfers. From 2013 onward, blastocysts were transferred on Day 5. If Day 5 was a Sunday, the transfer was delayed to Day 6. Univariable logistic regression analysis was performed to identify potential confounders. Factors with a P-value <0.1 were included in the multivariable logistic regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE: We included 334 single elective Day 5 and 268 elective Day 6 transfers. The unadjusted odds for implantation between Day 5 and Day 6 groups were 0.35 (95% CI 0.25-0.49). A univariable logistic regression analysis identified maternal age, BMI, cumulative FSH dose, number of cryopreserved embryos, score of inner cell mass and trophectoderm and day of transfer as predictors of clinical pregnancy. When adjusting for these variables in a multivariable logistic regression analysis, the implantation odds for Day 5 transfer remained significantly higher than Day 6 (OR 0.34; 95% CI 0.22-0.52). LIMITATIONS, REASONS FOR CAUTION: The study was conducted on good prognosis patients. The majority of Day 6 transfers were performed in the beginning of the study period. Day 5 transfers were generally performed in the end of the study period. This difference in time of recruitment may cause a minor variation in the data but a subanalysis indicates that this potential variation is negligible. Day 5 scores were higher in the Day 5 transfer group. WIDER IMPLICATIONS OF THE FINDINGS: Based on the findings in this study, transfers should be performed on Day 5. If Day 5 transfers are logistically impossible to perform, it is be preferable to cryopreserve the blastocyst and transfer in another cycle on Day 5, as Day 6 transfers should be avoided. STUDY FUNDING/COMPETING INTEREST(S): None.

Timing of human preimplantation embryonic development is confounded by embryo origin.

STUDY QUESTION: To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER: Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence. WHAT IS KNOWN ALREADY: Several studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations. STUDY DESIGN, SIZE, DURATION: Infertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Moderate intra-class correlation coefficients (0.16-0.31) were observed for all parameters except duration of the 3-cell stage, which demonstrates that embryos from one patient elicit clustering at a patient level. No single patient- and treatment-related factor was found to systematically influence the timing from cleavage to blastocyst stage, which indicates that no individual patient-related factor can be identified that separately explains the clustering throughout the entire developmental stages. The blastocyst parameters were more affected by patient-related factors than cleavage stage parameters, as tEB occurred significantly later with older age (0.29 h/year (95% confidence interval: CI 0.03; 0.56)), while both tEB and tFB occurred significantly later with increasing dose of FSH (tEB: 0.12 h/100 IU FSH (95% CI 0.01;0.24); tFB 0.14 h/100 IU FSH (95% CI 0.03;0.27)) and with more previous attempts (tEB: 1.2 h/attempt (95% CI 0.01;2.5); tFB 1.4 h/attempt (0.10;2.7)). Fertilization method affected timing of the first division, with ICSI embryos cleaving significantly faster than IVF embryos (-3.6% (95% CI -6.4; -0.77)), whereas no difference was found in the subsequent divisions. The univariable regression analysis identified female age, cumulative FSH dose, degree of blastocyst expansion, score of the inner cell mass and timing of full blastocyst formation as predictors of live birth. The timing of full blastocyst formation (tFB) did not remain significant when adjusting for age, number of previous cycles and cumulative FSH dose, which were the parameters shown to influence tFB in the mixed regression model. LIMITATIONS, REASONS FOR CAUTION: Only good prognosis patients were enrolled, so these results may not be generalized to all infertile women. Not all patient-related factors were investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline the importance of treating embryos as dependent observations and suggest a high risk of patient-based confounding in retrospective studies. The impact of confounders and the embryo origin needs to be addressed in order to apply appropriate statistical models in observational studies. Furthermore, this observation emphasizes the need for RCTs for evaluating use of time-lapse parameters for embryo selection. STUDY FUNDING/COMPETING INTERESTS: Funding for the cohort study was provided by the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and NordicInfu Care research grant. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from MSD and Ferring. K.K. is funded by a grant from the Danish Council for Independent Research Medical Sciences. The authors declare no competing interest.

Cervical conization doubles the risk of preterm and very preterm birth in assisted reproductive technology twin pregnancies.

STUDY QUESTION: Does cervical conization add an additional risk of preterm birth (PTB) in assisted reproduction technology (ART) singleton and twin pregnancies? SUMMARY ANSWER: Cervical conization doubles the risk of preterm and very PTB in ART twin pregnancies. WHAT IS KNOWN ALREADY: ART and cervical conization are both risk factors for PTB. STUDY DESIGN, SIZE, DURATION: In this national population-based controlled cohort study, we included all ART singletons and twin deliveries from 1995 to 2009 in Denmark by cross-linkage of maternal and child data from the National IVF register and the Medical Birth register. Furthermore, control groups of naturally conceived (NC) singletons and twins were extracted. Cervical diagnoses were obtained from the Danish Pathology register. Cervical conization included both cold knife cone and LEEP (loop electrosurgical excision procedure) but not cervical biopsies. The main outcomes measures were PTB (PTB ≤ 37 + 0 gestational weeks), very preterm birth (VPTB ≤ 32 + 0 gestational weeks) and preterm premature rupture of membranes (PPROM). PARTICIPANTS/MATERIALS, SETTING, METHODS: In all 16 923 ART singletons and 4829 ART twin deliveries were included. A random sample of NC singletons, 2-fold the size of the ART singleton group matched by date and year of birth (n = 33 835) and all NC twin deliveries (n = 15 112), was also extracted. Multiple logistic regression analyses were performed to adjust for the following confounders: maternal age, parity, year of child birth and sex of child. MAIN RESULTS AND THE ROLE OF CHANCE: Cervical morbidity (dysplasia and conization) was more often observed in ART pregnancies (6.2% of ART singletons and 5.4% ART twins) than in NC pregnancies (4.2% for NC singletons and 4.5% for NC twins), both for singletons and twins. In ART singleton deliveries, the PTB rate was 13.1 versus 8.2% in women with and without conization, respectively, with an adjusted odds ratio (aOR) of 1.56 [95% confidence interval (CI) 1.21-2.01]. In ART twin deliveries, the prevalence of PTB was 58.2 versus 41.3% in women with and without conization, respectively, with an aOR 1.94 (95% CI 1.36-2.77), and the risk of VPTB was also doubled. Furthermore, previous dysplasia (without conization) increased the risk of VPTB in ART twins (aOR 1.74, 95% CI 1.04-2.94). Cervical dysplasia did not increase the risk of any of the other adverse outcomes in ART singletons or twins. The risk of PPROM was increased in both in ART and NC singleton deliveries with conization versus no conization; however, this increased risk of PPROM after conization was not observed in either ART or NC twin pregnancies. LIMITATIONS, REASONS FOR CAUTION: We were not able to adjust for the height of the cervical cone or the severity of the cervical intraepithelial neoplasia (CIN) or the time window between diagnosis of CIN and ART treatment. The finding on an increased risk of VPTB in ART twin pregnancies after dysplasia without conization may be random as we found no other increased risk after dysplasia alone either in singletons or in twins. WIDER IMPLICATIONS OF THE FINDINGS: After ART and prior conization, 58% of twin pregnancies versus 13% of ART singleton pregnancies result in PTB. There is a doubled risk of preterm delivery in ART twins with conization versus ART twins with no prior conization. Single-embryo transfer should always be recommended in women with prior conization irrespective of female age, embryo quality and prior number of ART attempts. STUDY FUNDING/COMPETING INTERESTS: No external funding was achieved for this project.

Nuclear magnetic resonance metabolomic profiling of Day 3 and 5 embryo culture medium does not predict pregnancy outcome in good prognosis patients: a prospective cohort study on single transferred embryos.

STUDY QUESTION: Does the metabolomic profile, obtained with nuclear magnetic resonance (NMR), of spent culture media from human embryos correlate with reproductive potential in a cohort of good prognosis patients? SUMMARY ANSWER: In a large cohort of single transferred blastocysts from a homogeneous group of good prognosis patients, we find a high degree of individual variation in the metabolome that, however, has no relation to pregnancy outcome. WHAT IS KNOWN ALREADY: Differences among various specific metabolites have been linked to reproductive potential. Although results from retrospective near infrared (NIR) spectroscopy analyses of spent culture medias from transferred embryos were promising, randomized controlled trials were unable to demonstrate that NIR analysis improved pregnancy rates. Therefore, a more detailed investigation of the relation between embryo metabolism and reproductive potential is required. NMR is a powerful technique that provides detailed structural and dynamic information. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted at the Fertility Clinic, Aarhus University Hospital between February 2011 and July 2012. Infertile patients aged <38 years without endometriosis were offered participation and their embryos were included if greater than or equal to eight oocytes were retrieved. In total, 161 infertile patients were included in the cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spent culture media was collected on Days 3 and 5 after oocyte retrieval from 148 single transferred embryos. NMR spectra were obtained from 12 µl of spent media. Data were quantitatively analysed using multivariate analysis with respect to pregnancy outcome, defined as a live fetus by ultrasound in gestational Week 8, along with patient and treatment related variables such as embryo score, age, BMI, fertilization method and cause of infertility. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 148 cycles were included in the analysis [embryo transfer cancelled (n = 12), no media collected (n = 1)]. Clinical pregnancy was confirmed in 47 patients (32%). We obtained high quality NMR spectra for 141 Day 3 and 137 Day 5 samples. Our spectra show a high degree of individual variation. Multivariate data analysis was performed on spectral data with several different pre-processing combinations, i.e. binning, alignment, normalization and scaling in the attempt to develop a valid prediction model. Different strategies of multivariate analysis showed, however, no correlation between the NMR profiles and pregnancy outcome, patient or treatment characteristics. No model could therefore be developed for prediction of pregnancy outcome. We conclude that within this group of good prognosis patients, large-scale metabolic variations between embryos detected with NMR have no apparent association with pregnancy outcome. LIMITATIONS, REASONS FOR CAUTION: Although this study is the largest we know of using NMR to investigate metabolomic profiles of single-transferred embryos, there may be differences that would be detected with a larger study. When analysing such a small sample volume, even small variations in the amount of media and dilution may introduce a large uncertainty in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our study questions the usefulness of the entire metabolome for embryo selection, which should direct the search for viability markers in the culture media towards individual components. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation. Research at the Fertility Clinic, Aarhus Universtity Hospital is supported by an unrestricted grant from MSD and Ferring. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: NCT01139268.

Time-lapse parameters as predictors of blastocyst development and pregnancy outcome in embryos from good prognosis patients: a prospective cohort study.

STUDY QUESTION: Do early time-lapse parameters predict which embryos will develop to high-quality blastocysts and does timing of development differ between embryos that implant and those that do not. SUMMARY ANSWER: Development to high-quality blastocysts could be predicted within the first 48 h of culture, whereas time-lapse parameters could not predict pregnancy. WHAT IS KNOWN ALREADY: Historical cohort studies on embryos from unselected groups of patients have suggested several putative kinetic markers of viability. Before well-designed randomized studies can be conducted, relevant selection models based on solid data must be developed. So far conclusions from the previous studies are ambiguous. STUDY DESIGN, SIZE, DURATION: A prospective cohort study conducted from February 2011 to June 2012. A total of 571 ICSI embryos from 92 patients were included in the blastocyst development analysis and 84 single embryo transfers were included in the pregnancy outcome analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos from women aged <38 years, with no endometriosis and ≥ 8 oocytes retrieved. University affiliated clinic. Embryos were cultured in a time-lapse incubator till Day 6. Logistic regression analysis was performed with variables selected based on indication. MAIN RESULTS AND THE ROLE OF CHANCE: Duration of the first cytokinesis, duration of the 3-cell stage and direct cleavage to 3-cells predicted development to high-quality blastocyst. We found no difference in timing between implanted and non-implanted embryos. LIMITATIONS, REASONS FOR CAUTION: A larger study might detect differences in timing between implanted and non-implanted embryos. The cohort consisted of good prognosis patients only and may not be representative of the entire IVF population. WIDER IMPLICATIONS OF THE FINDINGS: Our results in context with the lack of consistency in previous studies and the presumed influences of different external factors indicate that a universal algorithm for optimal timing of development might not be feasible. The apparent negative significance of division patterns that differ from the expected may imply that time-lapse will facilitate de-selection of embryos. STUDY FUNDING/COMPETING INTEREST(S): Funding for the present study was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and by an unrestricted grant from MSD and Ferring. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: The study was registered at ClinicalTrial.gov with accession number NCT01139268.

Stress, distress and outcome of assisted reproductive technology (ART): a meta-analysis.

BACKGROUND: A number of studies have investigated the relationship between psychological factors such as stress and distress (measured as anxiety and depression) and outcomes of assisted reproductive technology (ART). The results, however, are inconsistent, and the strength of any associations remains to be clarified. We conducted a systematic review and meta-analysis of the results of studies reporting on the associations between stress, anxiety, and depression and ART outcomes. METHODS: Prospective studies reporting data on associations between stress or distress in female patients and ART outcome were identified and evaluated by two independent researchers according to an a priori developed codebook. Authors were contacted in cases of insufficient data reporting. Stress was defined as perceived stress, work-related stress, minor life events or major life events, and distress was defined as anxiety or depression. RESULTS: A total of 31 prospective studies were included. Small, statistically significant, pooled effect sizes were found for stress [ESr, effect size correlation) = -0.08; P = 0.02, 95% confidence interval (CI): -0.15, -0.01], trait anxiety (ESr = -0.14; P = 0.02, 95% CI: -0.25, -0.03) and state anxiety (ESr = -0.10, P = 0.03, 95% CI: -0.19, -0.01), indicating negative associations with clinical pregnancy rates. A non-significant trend (Esr = -0.11, P = 0.06) was found for an association between depression and clinical pregnancy. For serum pregnancy tests and live birth rates, associations between trait anxiety or state anxiety were not significant. The fail safe number did not exceed the suggested criterion in any analyses, between-study heterogeneity was considerable and the mean age, mean duration of infertility and percentage of first time ART attenders in the study samples were found to moderate several of the associations. CONCLUSIONS: Small but significant associations were found between stress and distress and reduced pregnancy chances with ART. However, there were a limited number of studies and considerable between-study heterogeneity. Taken together, the influence of stress and distress on ART outcome may appear somewhat limited.

IVF and stillbirth: a prospective follow-up study.

BACKGROUND: Previous studies have indicated that the risk of stillbirth is increased in singleton pregnancies achieved after assisted reproduction technology (ART). However, no previous study fully accounted for factors with potential influence on the risk of stillbirth. Further, whether fertility treatment, the possible reproductive pathology of the infertile couples or other characteristics related to being subfertile may explain a possible association with stillbirth remains unclear. This study compares the risk of stillbirth in women pregnant after fertility treatment (IVF/ICSI and non-IVF ART) and subfertile women with that in fertile women. METHODS: We used prospectively collected data from the Aarhus Birth Cohort, Denmark and included information about 20 166 singleton pregnancies (1989-2006). Outcome measure was stillbirth. RESULTS: The risk of stillbirth in women who conceived after IVF/ICSI was 16.2 per thousand ( per thousand) and in women who conceived after non-IVF ART 2.3 per thousand. In fertile and subfertile women, the risk of stillbirth was 3.7 per thousand and 5.4 per thousand, respectively. Compared with fertile women, women who conceived after IVF/ICSI had more than four times the risk of stillbirth [odds ratio (OR): 4.44, 95% confidence interval (CI): 2.38-8.28], and adjustments for maternal age, BMI, education, smoking habits and alcohol and coffee intake during pregnancy had only minor impact on the findings (OR: 4.08; 95% CI: 2.11-7.93). The risk of stillbirth in women who conceived after non-IVF ART and in women who conceived spontaneously with a waiting time to pregnancy of a year or more was not significantly different from the risk in women with a shorter time to pregnancy. CONCLUSIONS: Compared with fertile women, women who conceived by IVF/ICSI had an increased risk of stillbirth that was not explained by confounding. Our results indicate that the increased risk of stillbirth seen after fertility treatment is a result of the fertility treatment or unknown factors pertaining to couples who undergo IVF/ICSI.

Stressful life events are associated with a poor in-vitro fertilization (IVF) outcome: a prospective study.

BACKGROUND: There is preliminary evidence to suggest an impact of stress on chances of achieving a pregnancy with in-vitro fertilization (IVF). The majority of the available research has focused on stress related to infertility and going through IVF-treatment, and it is still unclear whether non-fertility-related, naturally occurring stressors may influence IVF pregnancy chances. Our aim was to explore the association between IVF-outcome and negative, i.e. stressful, life-events during the previous 12 months. METHODS: Prior to IVF, 809 women (mean age: 31.2 years) completed the List of Recent Events (LRE) and questionnaires measuring perceived stress and depressive symptoms. RESULTS: Women who became pregnant reported fewer non-fertility-related negative life-events prior to IVF (Mean: 2.5; SD: 2.5) than women who did not obtain a pregnancy (Mean: 3.0; SD: 3.0) (t(465.28) = 2.390, P = 0.017). Logistic regression analyses revealed that the number of negative life-events remained a significant predictor of pregnancy (OR: 0.889; P = 0.02), when controlling for age, total number of life-events, perceived stress within the previous month, depressive symptoms, and relevant medical factors related to the patient or treatment procedure, including duration of infertility, number of oocytes retrieved and infertility etiology. Mediation analyses indicated that the association between negative life events and IVF pregnancy was partly mediated by the number of oocytes harvested during oocyte retrieval. CONCLUSION: A large number of life-events perceived as having a negative impact on quality of life may indicate chronic stress, and the results of our study indicate that stress may reduce the chances of a successful outcome following IVF, possibly through psychobiological mechanisms affecting medical end-points such as oocyte retrieval outcome.

[Preimplantation genetic diagnosis. The first experiences in Denmark]. / Praeimplantations genetisk diagnostik. De første erfaringer i Danmark.

INTRODUCTION: Preimplantation genetic diagnosis (PGD) is a possible alternative to prenatal diagnosis, whereby families with serious inherited diseases can avoid having children with the disease. The genetic diagnosis is performed on embryos before implantation and therefore implies IVF. Hence, PGD offers the possibility of transferring embryos without disease, thereby avoiding termination of pregnancy owing to an affected fetus. MATERIAL AND METHODS: Activities at the Centre for Preimplantation Genetic Diagnosis at Aarhus University Hospital since its opening in February 1999 are described. The fluorescent in situ hybridisation (FISH) technique was used for sex selection (hemophilia A and Duchenne's muscular dystrophy) and translocations. The polymerase chain reaction (PCR) was used for cystic fibrosis. RESULTS: Of 20 PGD cycles started, 15 were successful in terms of transference of healthy or carrier embryos. A positive pregnancy test was found after six of 15 embryo transfers (40%) with two subsequent clinical pregnancies. CONCLUSIONS: The present pregnancy rates with PGD are comparable to those following IVF; the clinical pregnancy rate may seem low, but the cycle numbers are small. Preimplantation genetic diagnosis seems to be a realistic alternative for selected genetic diseases, in cases where the couple find abortion unacceptable.

Serological investigation of Mycoplasma genitalium in infertile women.

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.

Multiplex PCR for screening of microdeletions on the Y chromosome.

PURPOSE: The aim of this study was to develop a multiplex PCR protocol, which could be suitable for screening of microdeletions in the three azoospermia factor (AZF) regions on the Y chromosome. METHODS: In the screening protocol, 36 known sequence tagged site (STS) primer pairs were first tested in single PCR reactions and thereafter combined in multiplex PCR to test for specificity and sensitivity in order to develop a stable and reliable multiplex PCR assay to detect Y microdeletions. RESULTS: Of the 36 primers tested, 11 turned out not to be specific or produced PCR products that were too weak, and they were therefore not used in the multiplex PCR. The remaining 25 STSs were selected on the basis of their ability to be reproducibly amplified with each other using identical amplification conditions. Five multiplex sets, each consisting of five primer pairs, were established in the multiplex PCR setup. CONCLUSION: The multiplex PCR protocol presented in this study is an easy and reliable method for detection of Y chromosome microdeletions and could be used for screening of infertile men to allow genetic counseling about the risk of transmitting infertility from father to son.

Friendly IVF: patient opinions.

BACKGROUND: The present trend towards low stimulation protocols in IVF calls for evaluation of patient attitudes. METHODS: This study compared results of a 23-item questionnaire mailed to 167 patients receiving a low stimulation type of regimen (LS-IVF) (unstimulated cycle or clomiphene) and to 116 patients treated by a standard protocol (S-IVF) (long-down regulation with gonadotrophin-releasing hormone analogue and FSH or human menopausal gonadotrophin). RESULTS: Around two-thirds of all responders in both groups deemed side-effects important, but side-effects and stress associated with hormone treatment were more prevalent in patients receiving S-IVF than LS-IVF. Stress due to cycle cancellation was acceptable, mild or not perceptible in significantly (P < 0.005) more patients receiving LS-IVF [48% (36/75)] compared with patients having S-IVF [26% (8/31)]. Of patients having tried the LS-IVF protocol, 93% (125/135) would suggest either LS-IVF or a sequence of this and S-IVF as a future treatment "package" compared with only 53% (33/63) in the S-IVF group (chi(2) = 43.08, P < 0.0001). The LS-IVF group showed a significant trend towards acceptance of higher number of treatment cycles. CONCLUSIONS: The patients seemed to prefer the simplicity and short duration of a low stimulation regimen in spite of drawbacks such as a high risk of cycle cancellations and accordingly the necessity for more treatment cycles.

A randomized study comparing IVF in the unstimulated cycle with IVF following clomiphene citrate.

The efficiency of IVF in unstimulated cycles was compared with that following ovarian stimulation with clomiphene citrate in a simple protocol with ultrasound monitoring only. A total of 132 couples with no previous IVF attempts, selected by female age <35 years, indication for intracytoplasmic sperm injection or infertility caused by tubal factor or unexplained infertility were randomized to the two protocols. Randomization yielded two comparable groups. The clomiphene group (68 couples) performed significantly better than the unstimulated group (64 couples) in terms of number of cycles with oocyte harvest (90/111 or 81% versus 65/114 or 57%; chi(2) = 9.21, P < 0.002), embryo transfers per started cycle (59/111 or 53% versus 29/114 or 25%; chi(2) = 18.14, P < 0.0001), live intrauterine pregnancy rate per started cycle (20/111 or 18% versus 4/114 or 4%; chi(2) = 12.42, P < 0.0001), live intrauterine pregnancy rate per embryo transfer (20/59 or 34% versus 4/29 or 14%; chi(2) = 3.96, P = 0.047), but not in terms of implantation rate (22/85 or 26% versus 4/29 or 14%; chi(2) = 1.65). Only two twin pregnancies occurred. Modest side-effects were recorded following clomiphene. Accordingly, a simple clomiphene citrate protocol, but not IVF in unstimulated cycles, seems compatible with the concept of 'friendly IVF', yielding a fair pregnancy rate both per cycle started and per embryo transfer in selected patients. The results do not substantiate any important negative anti-oestrogenic effects of clomiphene.

Assessment of the follicular cortisol:cortisone ratio.

Cortisol and cortisone concentrations in serum and follicular fluid (FF) from women undergoing in-vitro fertilization (IVF) treatment were monitored. Four groups were included: group 1, women in their natural menstrual cycle having an endogenous mid-cycle surge of gonadotrophins; group 2, women in their natural menstrual cycle receiving human chorionic gonadotrophin (HCG) for ovulation induction; group 3, women receiving exogenous gonadotrophins for ovarian stimulation and HCG for ovulation induction; and group 4, women receiving exogenous gonadotrophins for ovarian stimulation, follicles being aspirated immediately before administration of HCG. In this study, 12 follicles contained oocytes which resulted in clinical pregnancy after IVF. Cortisone concentrations were significantly higher in FF compared with that of matched serum samples, while the opposite was observed for cortisol, resulting in cortisol:cortisone ratios being significantly lower in FF compared with serum. FF from group 4 showed significantly higher cortisone concentrations than FF from each of the other three groups. FF from group 1 showed significantly higher cortisone concentrations and significantly lower cortisol:cortisone ratios in comparison with groups 2 and 3. None of the observed parameters pinpointed any of the follicles containing oocytes which resulted in a clinical pregnancy. The intrafollicular concentrations of cortisol and cortisone suggest that pre-ovulatory follicles actively convert cortisol to cortisone. Neither FF concentrations of cortisol and cortisone nor the cortisol:cortisone ratio seem to reflect implantation potential of the derived pre-embryos.