C.B. Lambalk

Comparison of inter- and intra-cycle variability of anti-Müllerian hormone and antral follicle counts

BACKGROUND The antral follicle count (AFC) and anti-Müllerian hormone (AMH) both represent age-related follicular decline quite accurately, although long-term follow-up studies are still lacking. The best ovarian reserve test would need only a single, cycle-independent measurement to be representative. METHODS To compare the inter- and intra-cycle stability of AFC and AMH, we used age-adjusted intra-class correlation coefficients (ICCs). To measure inter-cycle stability across a number of up to four menstrual cycles, we used data, prospectively collected for the purpose of an other study, from 77 regularly cycling, infertile women aged 24-40 years. AMH and AFC values were measured on cycle day 3. To study intra-cycle variability, we used data from a prospective cohort study of 44 regularly cycling volunteers, aged 25-46 years and measured AMH and assessed the AFC (2-10 mm) every 1-3 cycle days. RESULTS Between menstrual cycles, AFC and AMH varied between 0 and 25 follicles (median 10), and 0.3 and 27.1 ng/ml (median 4.64). The difference in age-adjusted ICC between AMH [ICC, 0.89 (95% CI, 0.84-0.94)] and AFC [ICC, 0.71 (95% CI, 0.63-0.77)] was 0.18 (95% CI, 0.12-0.27). For the intra-cycle variation, 0-43 antral follicles (median 7) were counted per volunteer. The difference in age-adjusted ICC between AMH [ICC, 0.87 (95% CI, 0.82-0.91)] and AFC [ICC, 0.69 (95% CI, 0.46-0.82)] was 0.18 (95% CI, 0.034-0.42). CONCLUSIONS Serum AMH demonstrated less individual intra- and inter-cycle variation than AFCs and may therefore be considered a more reliable and robust means of assessing ovarian reserve in subfertile women.

Added value of ovarian reserve testing on patient characteristics in the prediction of ovarian response and ongoing pregnancy: an individual patient data approach

BACKGROUND Although ovarian reserve tests (ORTs) are frequently used prior to IVF treatment for outcome prediction, their added predictive value is unclear. We assessed the added value of ORTs to patient characteristics in the prediction of IVF outcome. METHODS An individual patient data (IPD) meta-analysis from published studies was performed. Studies on FSH, anti-Müllerian hormone (AMH) or antral follicle count (AFC) in women undergoing IVF were identified and authors were contacted. Using random intercept logistic regression models, we estimated the added predictive value of ORTs for poor response and ongoing pregnancy after IVF, relative to patient characteristics. RESULTS We were able to collect 28 study databases, comprising 5705 women undergoing IVF. The area under the receiver-operating characteristic curve (AUC) for female age in predicting poor response was 0.61. AFC and AMH each significantly improved the model fit (P-value <0.001). Moreover, almost a similar accuracy was reached using AMH or AFC alone (AUC 0.78 and 0.76, respectively). Combining the two tests, however, did not improve prediction (AUC 0.80, P = 0.19) of poor response. In predicting ongoing pregnancy after IVF, age was the best single predictor (AUC 0.57), and none of the ORTs added any value. CONCLUSIONS This IPD meta-analysis demonstrates that AFC and AMH clearly add to age in predicting poor response. As single tests, AFC and AMH both fully cover the prediction of poor ovarian response. In contrast, none of the ORTs add any information to the limited capacity of female age to predict ongoing pregnancy after IVF. The clinical usefulness of ORTs prior to IVF will be limited to the prediction of ovarian response.

Anti-Müllerian Hormone, Inhibin B, and Antral Follicle Count in Young Women with Ovarian Failure

CONTEXT Ovarian dysfunction is classically categorized on the basis of cycle history, FSH, and estradiol levels. Novel ovarian markers may provide a more direct insight into follicular quantity in hypergonadotropic women. OBJECTIVE The objective of the study was to investigate the distribution of novel ovarian markers in young hypergonadotropic women as compared with normogonadotropic regularly menstruating women. DESIGN This was a nationwide prospective cohort study. SETTING The study was conducted at 10 hospitals in The Netherlands. PATIENTS Women below age 40 yr with regular menses and normal FSH (controls; n = 83), regular menstrual cycles and elevated FSH [incipient ovarian failure (IOF); n = 68]; oligomenorrhea and elevated FSH [referred to as transitional ovarian failure (TOF); n = 79]; or at least 4 months amenorrhea together with FSH levels exceeding 40 IU/liter [premature ovarian failure (POF); n = 112]. MAIN OUTCOME MEASURES Serum levels of anti-Müllerian hormone (AMH), inhibin B, and antral follicle count (AFC) was measured. RESULTS All POF patients showed AMH levels below the fifth percentile (p(5)) of normoovulatory women. Normal AMH levels (>p(5)) could be identified in 75% of IOF, 33% of TOF patients, and 98% of controls. AFC and AMH levels changed with increasing age (P < 0.0001), whereas inhibin B did not (P = 0.26). AMH levels were significantly different between TOF and IOF over the entire age range, whereas AFC became similar for TOF and IOF at higher ages. CONCLUSIONS Compared with inhibin B and AFC, AMH was more consistently correlated with the clinical degree of follicle pool depletion in young women presenting with elevated FSH levels. AMH may provide a more accurate assessment of the follicle pool in young hypergonadotropic patients, especially in the clinically challenging subgroups of patients with elevated FSH and regular menses (i.e. IOF) and in hypergonadotropic women with cycle disturbances not fulfilling the POF diagnostic criteria (i.e. TOF).

Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection

BACKGROUND The morphology of an embryo has a limited predictive value for assessing viability and ongoing pregnancy, therefore new selection tools are needed to maintain success rates with single-embryo transfer (SET). In this study, we investigated if metabolomic profiling of biomarkers of embryo culture medium by near-infrared (NIR) spectroscopy has a correlation with ongoing pregnancy in SET. METHODS A total of 333 patients scheduled for in vitro fertilization (IVF) with SET were included in the study. Embryos were selected for transfer by morphological criteria on Days 2 and 3 of in vitro culture, and left over culture media samples were analyzed by NIR spectroscopy. RESULTS The NIR spectral analysis produced unique metabolomic profiles that correlated to an embryos reproductive potential. Resulting relative viability scores between positive and negative pregnancy outcomes were statistically significant (P < 0.03). A logistic regression of factors correlated to pregnancy outcomes showed that maternal age, percent fragmentation and relative viability scores all demonstrated a relationship. The extent of the correlation was determined by accuracy computation, where the accuracy of assessing viable embryos on Day 3 by metabolomic profiling was 53.6% and the accuracy of the morphological selection was 38.5%. In addition, the positive predictive value of metabolomic profiling was 0.365 and the negative predictive value was 0.830. CONCLUSIONS NIR metabolomic profiling of spent embryo culture media was able to distinguish viable embryos from non-viable embryos for reproduction.

Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer

OBJECTIVE To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. DESIGN Retrospective study. SETTING Academic and a private assisted reproductive technology (ART) programs. PATIENT(S) Women undergoing single embryo transfer after IVF. INTERVENTION(S) Spent embryo culture media were collected after single embryo transfer on day 3 (n = 304) or day 2 (n = 181) and analyzed by near infrared spectroscopy. Near infrared spectral regions were correlated to reproductive potential using a genetic algorithm optimization. Models of these spectral regions were used to calculate viability indices, and were validated by blinded analysis of a subset (n = 60) of samples. Implantation rates were also compared between embryos of higher (>or=0.3) and lower (<0.3) viability indices, and within each morphology grade. MAIN OUTCOME MEASURE(S) Viability index and embryo viability. RESULT(S) Mean viability indices of embryos that resulted in positive fetal cardiac activity were significantly higher compared with embryos that did not for both day 2 and day 3 embryos. Blinded validation of the day 2 model proved to be significant. Increasing viability index values correlated with an increase in pregnancy. Viability indices were found to be independent of morphology for both day 2 and day 3 embryos. Implantation rates were significantly higher among embryos with viability indices >or=0.3. CONCLUSION(S) Metabolomic profiling of human embryo culture media using near infrared spectroscopy is independent of morphology and correlates with reproductive potential of embryos.

Testing ovarian reserve to predict age at menopause

In modern society with women delaying pregnancy, predicting the age of the natural menopause with its preceding infertility will allow making informed choices about when to try starting to have children. Also if premature menopause could be predicted in young women, strategies could be instigated to reduce the long term health risks of early estrogen deficiency. This review examines the physiology of ovarian ageing, with the menopause being the final outcome. Long and short term predictive markers of the age of the menopause and the preceding natural infertility are evaluated. Many subtle changes in the endocrine regulation of ovarian function with advancing age may seem interesting but currently are not clinically useful as a predictive test. Examples are changes in concentrations of estradiol, progesterone, luteinizing hormone (LH) and activin, as well as follicle dynamics. Other features hold more promise. Among these are chronological age, family history, anti-Müllerian hormone (AMH), poor response to in vitro fertilization (IVF), basal follicle-stimulating hormone (FSH) and the antral follicle count for long term prediction. For short term prediction, cycle shortening and occurrence of vasomotor symptoms may prove useful. To date, none of these markers has been found to have sufficient predictive accuracy in individual women. Results of new and ongoing longitudinal studies may provide better predictive models. In particular, use of genetic profiles may add to the accuracy of currently known markers.

Genome-wide association study in premature ovarian failure patients suggests ADAMTS19 as a possible candidate gene

BACKGROUND Spontaneous premature ovarian failure (POF) occurs in 1% of women and has major implications for their fertility and health. Besides X chromosomal aberrations and fragile X premutations, no common genetic risk factor has so far been discovered in POF. Using high-density single nucleotide polymorphism (SNP) arrays, we set out to identify new genetic variants involved in this condition. METHODS A genome-wide association study involving 309 158 SNPs was performed in 99 unrelated idiopathic Caucasian POF patients and 235 unrelated Caucasian female controls. A replication study on the most significant finding was performed. We specifically focused on chromosomal areas and candidate genes previously implicated in POF. RESULTS Suggestive genome-wide significant association was observed for rs246246 (allele frequency P = 6.0 x 10(-7)) which mapped to an intron of ADAMTS19, a gene known to be up-regulated in the female mouse gonads during sexual differentiation. However, replication in an independent Dutch cohort (60 POF patients and 90 controls) could not confirm a clear association (P = 4.1 x 10(-5) in a joint analysis). We did not observe strong evidence for any of 74 selected POF candidate genes or linkage regions being associated with idiopathic POF in Caucasian females, although suggestive association (P < 0.005) was observed for SNPs that mapped in BDNF, CXCL12, LHR, USP9X and TAF4B. CONCLUSION We observed a possible association between POF and a SNP in a biologically plausible candidate gene. Although limited by sample size, this proof-of-principle studys findings reveal ADAMTS19 as a possible candidate gene for POF and thus a larger follow-up study is warranted.

Comparison of ovarian function markers in users of hormonal contraceptives during the hormone-free interval and subsequent natural early follicular phases

OBJECTIVE The aim of this study was to evaluate whether values of FSH, LH, estradiol, anti-Müllerian hormone (AMH), inhibin B, antral follicle count (AFC) and ovarian volume (OV) determined on day 7 of the hormone-free interval are similar to values measured on days 2-5 of two subsequent natural menstrual cycles. In addition, values measured on day 7 of the hormone-free interval were examined for the purpose of predicting values measured on days 2-5 during the second natural cycle. METHODS In this study, 25 women using hormonal contraception provided a blood sample and underwent transvaginal ultrasound measurements on day 7 of the hormone-free interval and on cycle days 2-5 of two subsequent natural cycles. Changes were compared by repeated measures ANOVA and multivariate linear regression was used for prediction purposes. RESULTS Mean (SD) age of the participants was 26.3 (6.2) years. Overall significant decreases in FSH and inhibin B and significant increases in AMH, AFC and ovarian volume values were measured after discontinuation of hormonal contraception (P < 0.001, P = 0.04, P = 0.01, P < 0.001 and P = 0.004, respectively). Significant changes occurred both from day 7 of the hormone-free interval to natural cycle 1 as well as from natural cycle 1 to natural cycle 2. FSH, AMH and AFC values measured during days 2-5 of natural cycle 2 could be predicted by the corresponding values measured on day 7 of the hormone-free interval. CONCLUSION Hormonal and ultrasound markers of ovarian function in hormonal contraception users measured at the end of the hormone-free interval do not seem to represent subsequent natural early follicular phase values. However, these values can, in some cases (FSH, AMH and AFC), be used to predict early follicular phase values using calculated prediction equations, which need to be validated in future research.

Day 3 embryo selection by metabolomic profiling of culture medium with near-infrared spectroscopy as an adjunct to morphology: a randomized controlled trial

STUDY QUESTION Is the selection of a single Day 3 embryo by metabolomic profiling of culture medium with near-infrared (NIR) spectroscopy as an adjunct to morphology able to improve live birth rates in IVF, compared with embryo selection by morphology alone? SUMMARY ANSWER The live birth rate after embryo selection by NIR spectroscopy and morphology is not significantly different compared with the live birth rate after embryos were selected by morphology alone. WHAT IS KNOWN ALREADY The elevated incidence of pregnancy and neonatal problems associated with a high-twinning rate after IVF can only be successfully reduced by the transfer of one embryo. Current embryo assessment methods are unable to accurately predict the reproductive potential of an individual embryo. Today, a number of techniques are said to be more accurate at selecting the best embryo. One of these new technologies is metabolomic profiling of spent embryo culture media with the use of NIR spectroscopy. STUDY DESIGN, SIZE AND DURATION A double-blind, randomized controlled trial was conducted between 2009 and 2011, and included 417 couples undergoing IVF with a single embryo transfer. Randomization was performed centrally just before Ovum Pick-Up (OPU), using a computerized randomization program. Both patient and physician were unaware of the treatment allocation. To ensure blinding, the allocations were placed in consecutively numbered, opaque envelopes. Patients were randomized (1:1) into either the control group (embryo selection by morphology only) or the treatment group (embryo selection by morphology plus NIR spectroscopy of embryo culture medium). PARTICIPANTS/MATERIALS, SETTING AND METHODS At OPU, 208 patients were randomized to the morphology only group and 209 patients were randomized to the morphology plus viability score group. On Day 3, 163 patients in the control group and 146 patients in the treatment group met the inclusion criteria. The study was conducted in an academic hospital with IVF laboratory and three non-academic hospitals. MAIN RESULTS AND THE ROLE OF CHANCE Patient demographics and baseline characteristics were distributed equally over the two groups, except for embryo fragmentation, which was significantly higher in the treatment group. In the intention to treat analysis, the live birth rates were 31.7 and 26.8% for the control group and the treatment group, respectively (relative risk 0.84; 95% confidence interval 0.63-1.14, P=0.27). In the per protocol analysis, the live birth rates were 31.3 and 29.5% for the control group and the treatment group, respectively (relative risk 0.94; 95% confidence interval 0.67-1.32, P=0.73). For the treatment group, the embryological technicians independent choice (by morphology) of which embryo to transfer was recorded 138 times. In 75.4% (104 of 138) of the transfers, the embryo with the best morphology did not have the highest viability score. The live birth rate of these 104 transferred embryos was 30.8%. LIMITATIONS, REASONS FOR CAUTION A possible limitation of our study is the pre-selection of all embryos by morphology and dividing the cohort of available embryos into two groups: good quality embryos and poor quality embryos. As a consequence, we have probably selected for a better prognosis patient group. WIDER IMPLICATIONS OF THE FINDINGS To avoid the use of incompetent embryo selection tools at the expense of the patient, an evidence-based proof of clinical usefulness is essential before the implementation of new diagnostic tools in IVF laboratories. TRIAL REGISTRATION NUMBERS Dutch Trial Registry, registry number NTR1178.

Reproductive hormone concentrations in pregnancy and neonates: a systematic review.

Although much research focuses on hormones during gestation, little is known about the actual hormone concentrations within the fetal surroundings. The aim of this study was to combine all available oestrogen, androgen, sex hormone-binding globulin (SHBG), anti-Müllerian hormone (AMH), inhibin, gonadotrophin and dehydroepiandrosterone sulphate (DHEAS) concentrations during gestation and post partum into graphical representations reporting weighted mean hormone values. A systematic search was performed in Pubmed and Embase from inception to March 2012. Studies were evaluated by two reviewers; manuscripts were included if the actual hormone concentrations were reported together with the gestational age at time of sampling. A total of 97 articles were found eligible for this review. Maternal serum oestrogens, inhibin A, SHBG, androstenedione and testosterone rise during gestation, which is followed by a rapid decline in the post-partum period. For AMH and DHEAS, an inverse relationship is found, while gonadotrophin concentrations are negligible during gestation. For girls cord blood oestriol and post-partum FSH concentrations are higher, while for boys cord blood FSH and neonatal testosterone, inhibin B, LH and AMH concentrations are higher. In conclusion, longitudinally measured endocrine data during gestation and in the peri- and post-natal period are lacking, especially for twin pregnancies.