Luciano G. Nardo

Circulating basal anti-Müllerian hormone levels as predictor of ovarian response in women undergoing ovarian stimulation for in vitro fertilization.

OBJECTIVE To evaluate the clinical value of basal anti-Müllerian hormone (AMH) measurements compared with other available determinants, apart from chronologic age, in the prediction of ovarian response to gonadotrophin stimulation. DESIGN Prospective cohort study. SETTING Tertiary referral center for reproductive medicine and an IVF unit. PATIENT(S) Women undergoing their first cycle of controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). MATERIALS AND METHODS Basal levels of FSH and AMH as well as antral follicle count (AFC) were measured in 165 subjects. All patients were followed prospectively and their cycle outcomes recorded. MAIN OUTCOME MEASURE(S) Predictive value of FSH, AMH, and AFC for extremes of ovarian response to stimulation. RESULT(S) Out of the 165 women, 134 were defined as normal responders, 15 as poor responders, and 16 as high responders. Subjects in the poor response group were significantly older then those in the other two groups. Anti-Müllerian hormone levels and AFC were markedly raised in the high responders and decreased in the poor responders. Compared with FSH and AFC, AMH performed better in the prediction of excessive response to ovarian stimulation-AMH area under receiver operating characteristic curve (ROC(AUC)) 0.81, FSH ROC(AUC) 0.66, AFC ROC(AUC) 0.69. For poor response, AMH (ROC(AUC) 0.88) was a significantly better predictor than FSH (ROC(AUC) 0.63) but not AFC (ROC(AUC) 0.81). AMH prediction of ovarian response was independent of age and PCOS. Anti-Müllerian hormone cutoffs of >3.75 ng/mL and <1.0 ng/mL would have modest sensitivity and specificity in predicting the extremes of response. CONCLUSION(S) Circulating AMH has the ability to predict excessive and poor response to stimulation with exogenous gonadotrophins. Overall, this biomarker is superior to basal FSH and AFC, and has the potential to be incorporated in to work-up protocols to predict patients ovarian response to treatment and to individualize strategies aiming at reducing the cancellation rate and the iatrogenic complications of COH.

The effect of surgical treatment for endometrioma on in vitro fertilization outcomes: a systematic review and meta-analysis

OBJECTIVE To investigate the effect of surgical treatment of endometrioma on pregnancy rate and ovarian response to gonadotrophin stimulation in women undergoing IVF. DESIGN A systematic review and meta-analysis. SETTING Tertiary referral center for reproductive medicine. PATIENT(S) Subfertile women with endometrioma undergoing IVF. INTERVENTION(S) Surgical removal of endometrioma or expectant management. MAIN OUTCOME MEASURE(S) Clinical pregnancy rate and ovarian response to gonadotrophins (number of gonadotrophin ampoules, peak E(2) levels, number of oocytes retrieved, and number of embryos available for transfer). RESULT(S) A search of three electronic databases for articles published between January 1985 and November 2007 yielded 20 eligible studies. Meta-analysis was conducted for five studies that compared surgery vs. no treatment of endometrioma. There was no significant difference in clinical pregnancy rate between the treated and the untreated groups. Similarly, no significant difference was found between the two groups with regard to the outcome measures used to assess the response to controlled ovarian hyperstimulation with gonadotrophins. CONCLUSION(S) Collectively the available data in the literature show that surgical management of endometriomas has no significant effect on IVF pregnancy rates and ovarian response to stimulation compared with no treatment. Randomized controlled trials are needed before producing best-practice recommendations on this topic.

Anti-Müllerian hormone: poor assay reproducibility in a large cohort of subjects suggests sample instability

STUDY QUESTION What is the variability of anti-Müllerian hormone (AMH) concentration in repeat samples from the same individual when using the Gen II assay and how do values compare to Gen I [Diagnostic Systems Ltd (DSL)] assay results? SUMMARY ANSWER The Gen II AMH assay displayed appreciable variability, which can be explained by sample instability. WHAT IS KNOWN ALREADY AMH is the primary predictor of ovarian performance and is used to tailor gonadatrophin dosage in cycles of IVF/ICSI and in other routine clinical settings. Thus, a robust, reproducible and sensitive method for AMH analysis is of paramount importance. The Beckman Coulter Gen II ELISA for AMH was introduced to replace earlier DSL and Immunotech assays. The performance of the Gen II assay has not previously been studied in a clinical setting. STUDY DESIGN, SIZE AND DURATION We studied an unselected group of 5007 women referred for fertility problems between 1 September 2008 and 25 October 2011; AMH was measured initially using the DSL AMH ELISA and subsequently using the Gen II assay. AMH values in the two assays were compared using a regression model in log(AMH) with a quadratic adjustment for age. Additionally, women (n = 330) in whom AMH had been determined in different samples using both the DSL and Gen II assays (paired samples) identified and the difference in AMH levels between the DSL and Gen II assays was estimated using the age-adjusted regression analysis. A subset of 313 women had repeated AMH determinations (n = 646 samples) using the DSL assay and 87 women had repeated AMH determinations using the Gen II assay (n = 177 samples) were identified. A mixed effects model in log(AMH) was utilized to estimate the sample-to-sample (within-subject) coefficients of variation of AMH, adjusting for age. Laboratory experiments including sample stability at room temperature, linearity of dilution and storage conditions used anonymized samples. MAIN RESULTS AND THE ROLE OF CHANCE In clinical practice, Gen II AMH values were ∼20% lower than those generated using the DSL assay instead of the 40% increase predicted by the kit manufacturer. Both assays displayed high within-subject variability (Gen II assay CV = 59%, DSL assay CV = 32%). In the laboratory, AMH levels in serum from 48 subjects incubated at RT for up to 7 days increased progressively in the majority of samples (58% increase overall). Pre-dilution of serum prior to assay, gave AMH levels up to twice that found in the corresponding neat sample. Pre-mixing of serum with assay buffer prior to addition to the microtitre plate gave higher readings (72% overall) compared with sequential addition. Storage at -20°C for 5 days increased AMH levels by 23% compared with fresh samples. The statistical significance of results was assessed where appropriate. LIMITATIONS, REASONS FOR CAUTION The analysis of AMH levels is a retrospective study and therefore we cannot entirely rule out the existence of differences in referral practices or changes in the two populations. WIDER IMPLICATIONS OF THE FINDINGS Our data suggests that AMH may not be stable under some storage or assay conditions and this may be more pronounced with the Gen II assay. The published conversion factors between the Gen II and DSL assays appear to be inappropriate for routine clinical practice. Further studies are urgently required to confirm our observations and to determine the cause of the apparent instability. In the meantime, caution should be exercised in the interpretation of AMH levels in the clinical setting. CONFLICT OF INTEREST/STUDY FUNDING S. Roberts is supported by the NIHR Manchester Biomedical Research Centre.

The risk of monozygotic twins after assisted reproductive technology: a systematic review and meta-analysis

BACKGROUND It is estimated that there is at least a 2-fold rise in the incidence of monozygotic twinning after assisted reproductive technology compared with natural conception. This can result in adverse pregnancy outcomes. METHODS We searched MEDLINE, EMBASE and SCISEARCH for studies that estimated the risk of monozygotic twinning and its association with any particular assisted reproductive technique. Monozygotic twinning was defined by ultrasound or Weinberg criteria. A meta-analysis of the proportion of monozygotic twins was performed using both fixed and random effects models. RESULTS The search revealed 37 publications reporting on the incidence of monozygotic twins after assisted reproductive techniques. Twenty-seven studies met the inclusion criteria and were included in the meta-analysis. The summary incidence of monozygotic twins after assisted conception was 0.9% (0.8-0.9%). The incidence of monozygotic twins in natural conception is 0.4%. Blastocyst transfer and intracytoplasmic sperm injection are associated with 4.25 and 2.25 times higher risk of monozygotic twins. CONCLUSIONS The risk of monozygotic twins in assisted conception is 2.25 times higher than the natural conceptions. Larger studies reporting on monozygotic twinning following single-embryo transfer or after post-natal confirmation of zygosity with DNA analysis are warranted before definitive conclusions can be drawn and guidelines produced. In order to provide adequate pre-conceptional counselling, it is important to monitor the incidence of monozygotic twins in both natural and assisted conceptions. We suggest building a national multiple pregnancy database based on accurate diagnosis of zygosity.

The relationships between AMH, androgens, insulin resistance and basal ovarian follicular status in non-obese subfertile women with and without polycystic ovary syndrome

BACKGROUND Hyperandrogenaemia and insulin resistance are prominent features of polycystic ovary syndrome (PCOS) and influence the process of folliculogenesis in women with the endocrinopathy. Anti-Müllerian hormone (AMH) levels are elevated in women with PCOS and studies including IVF subjects have shown that this is a reliable marker of ovarian performance. The aims of this prospective study were to assess the relationship between insulin resistance, androgens and AMH, and whether AMH contributes to altered folliculogenesis in non-obese women with PCOS. METHODS A total of 232 IVF candidates, 49 of whom had PCOS according to the Rotterdam 2003 consensus criteria, were recruited. AMH levels and ovarian morphology were assessed. The relationships between AMH and insulin resistance and androgenaemia in patients with and without PCOS were studied. RESULTS PCOS patients were slightly older than controls (median ages 34 and 30 years, respectively). AMH generally increased with antral follicle count (AFC), insulin, homeostatic model assessment of tissue insulin sensitivity (HOMA-IR), testosterone, free androgen index and luteinising hormone, and decreased with chronological age, homeostatic model assessment of steady state beta cell function (HOMA-B) and serum sex hormone binding globulin (SHBG). For these relationships there were no significant differences in the slopes between PCOS and non-PCOS patients. The ratio of AMH per antral follicle (AMH/AF) was higher in PCOS patients. Both PCOS and non-PCOS groups showed a very similar increase in AMH with increases in AFC, but the PCOS patients had consistently higher AMH across all AFC levels. CONCLUSIONS These observations indicate that AMH is similarly related to insulin resistance and androgens in women with and without PCOS. This effect appears to be independent of age although an indirect causal effect due to ageing or some other mechanism cannot be ruled out. Excessive granulosa cell activity may be implicated in the abnormal follicular dynamic of the syndrome.

Anti-Müllerian hormone-tailored stimulation protocols improve outcomes whilst reducing adverse effects and costs of IVF

BACKGROUND Anti-Müllerian hormone (AMH) is increasingly used to quantify ovarian reserve, but it has not yet realized its full clinical potential in assisted reproduction technology. We investigated the possible benefits of using novel, stratified ovarian hyperstimulation protocols, tailored to individual AMH levels, compared with conventional stimulation. METHODS Retrospective data were collected from 769 women (first cycle of IVF, using fresh embryos), in a UK tertiary care unit: 346 women using conventional stimulation protocols; 423 women treated under new AMH-tailored protocols. RESULTS Embryo transfer rates increased significantly (79-87%: P= 0.002) after the introduction of AMH-tailored stimulation protocols. Pregnancy rate per cycle started and live birth rate also increased significantly compared with conventionally treated women (17.9-27.7%, P= 0.002 and 15.9-23.9%, P = 0.007, respectively). Moreover, in the AMH group, the incidence of the ovarian hyperstimulation syndrome (OHSS) fell significantly (6.9-2.3%, P = 0.002) and failed fertilization fell from 7.8 to 4.5%. The cost of fertility drug treatment fell by 29% per patient and the overall cost of clinical management of OHSS fell by 43% in the AMH group. GnRH antagonist protocols, introduced as part of AMH-tailored treatment, may have contributed to the observed improvements: however, within the AMH-tailored group, the live birth rate was not significantly different between agonist and antagonist-treated groups. CONCLUSIONS Although large, prospective, multicentre studies are indicated, we have clearly demonstrated that individualized, AMH-guided, controlled ovarian hyperstimulation protocols significantly improved positive clinical outcomes, reduced the incidence of complications and reduced the financial burden associated with assisted reproduction.

The likelihood of live birth and multiple birth after single versus double embryo transfer at the cleavage stage: a systematic review and meta-analysis

OBJECTIVE To determine whether a policy of elective single-embryo transfer (e-SET) lowers the multiple birth rate without compromising the live birth rate. DESIGN Systematic review and meta-analysis. SETTING Tertiary referral center for reproductive medicine and IVF unit. PATIENT(S) None. INTERVENTION(S) Searches of the Cochrane Controlled Trials Register, Meta-register for Randomized Controlled Trials (RCTs), EMBASE, MEDLINE, and SCISEARCH with no limitation on language and publication year, 1974 to 2008. SELECTION CRITERIA randomized, controlled trials comparing e-SET with double-embryo transfer (DET) for live birth and multiple birth rates after in vitro fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI). Nonrandomized trials and studies that included only patients who had blastocyst transfer were excluded. MAIN OUTCOME MEASURE(S) The likelihood of live birth per patient and multiple birth per total number of live births. Other outcomes included implantation rate, pregnancy rate, miscarriage and ectopic pregnancy rates, clinical pregnancy rate, ongoing pregnancy rate per patient, and preterm delivery rate per live birth. RESULT(S) Six trials (n=1354 patients) were included in the meta-analysis. Compared with DET, the e-SET policy was associated with a statistically significant reduction in the probability of live birth (RR 0.62; 95% CI, 0.53-0.72) and multiple birth (RR 0.06; 95% CI, 0.02-0.18). CONCLUSION(S) Elective-SET of embryos at the cleavage stage reduces the likelihood of live birth by 38% and multiple birth by 94%. Evidence from randomized, controlled trials suggests that increasing the number of e-SET attempts (fresh and/or frozen) results in a cumulative live birth rate similar to that of DET. Offering subfertile women three cycles of IVF will have a major impact on the uptake of an e-SET policy.

Anti-Müllerian hormone levels and antral follicle count in women enrolled in in vitro fertilization cycles : Relationship to lifestyle factors, chronological age and reproductive history

The aims of this prospective study were to investigate the relationship between anti-Müllerian hormone (AMH) and antral follicle count (AFC), and to determine whether these markers of ovarian reserve correlate with lifestyle factors, ethnicity, chronological age and reproductive history. Participants were 136 normo-ovulatory women undergoing infertility work-up within 3 months of their first ovarian stimulation cycle for in vitro fertilization. On day 3 of a spontaneous menstrual cycle, a blood sample for measurement of plasma AMH levels was taken and a transvaginal ultrasound scan to determine the AFC (follicles measuring 2–5 mm in diameter) was performed. Information about smoking, body mass index, alcohol consumption, ethnic origin, chronological age, age at menarche, years since menarche and gravidity were recorded using a case report form. The main outcome measures were plasma AMH concentrations and total number of small antral follicles (AFC). Median plasma levels of AMH were 2.0 ng/ml (interquartile range 1.1–3.6) and AFC was 10 (interquartile range 7–15). A positive correlation between AMH and AFC (r = 0.54, p < 0.0001) was found. AMH and AFC correlated negatively with age (r = −0.30, p < 0.001 and r = −0.27, p = 0.001 respectively) and number of years since menarche (r = −0.23, p = 0.007 and r = −0.21, p = 0.015 respectively), but not with any of the other measures. Circulating AMH levels and AFC correlated with each other and declined significantly with age. There were only weak, non-significant, correlations with lifestyle factors and reproductive history. These putative markers could be used individually or together to assess the age-related decline of ovarian function in normo-ovulatory candidates for IVF.

Age-related normograms of serum antimüllerian hormone levels in a population of infertile women: a multicenter study

OBJECTIVE To produce age-related normograms for serum antimüllerian hormone (AMH) level in infertile women without polycystic ovaries (non-PCO). DESIGN Retrospective cohort analysis. SETTING Fifteen academic reproductive centers. PATIENT(S) A total of 3,871 infertile women. INTERVENTION(S) Blood sampling for AMH level. MAIN OUTCOME MEASURE(S) Serum AMH levels and correlation between age and different percentiles of AMH. RESULT(S) Age-related normograms for the 3rd, 10th, 25th, 50th, 75th, 90th, and 97th percentiles of AMH were produced. We found that the curves of AMH by age for the 3rd to 50th percentiles fit the model and appearance of linear relation, whereas the curves of >75th percentiles fit cubic relation. There were significant differences in AMH and FSH levels and in antral follicle count (AFC) among women aged 24-33 years, 34-38 years, and ≥39 years. Multivariate stepwise linear regression analysis of FSH, age, AFC, and the type of AMH kit as predictors of AMH level shows that all variables are independently associated with AMH level, in the following order: AFC, FSH, type of AMH kit, and age. CONCLUSION(S) Age-related normograms in non-PCO infertile women for the 3rd to 97th percentiles were produced. These normograms could provide a reference guide for the clinician to consult women with infertility. However, future validation with longitudinal data is still needed.

The use of anti-Müllerian hormone and antral follicle count to predict the potential of oocytes and embryos

OBJECTIVE To investigate whether anti-Müllerian hormone (AMH) is better than antral follicle count (AFC) in predicting oocyte yield and embryo quality after controlled ovarian hyperstimulation for in vitro fertilization (IVF). STUDY DESIGN This is a prospective observational study involving 162 women (<40 years old) undergoing their first IVF cycle at an IVF unit within a university hospital. AMH and AFC measurements were made on day 3 of the cycle within 3 months of starting ovarian stimulation. A standard long down-regulation protocol using gonadotrophin releasing hormone agonist and recombinant follicle stimulating hormone was used. A maximum of two embryos were transferred on day 2 or 3 following oocyte retrieval. The primary outcome was the number of good quality embryos available for transfer and freezing. Embryos were graded according to the number of blastomeres, the difference in blastomere size and the degree of fragmentation, into grades 1-4. Secondary outcomes included the number of oocytes retrieved and fertilized and the live birth rate. Correlation between different parameters was calculated using Spearmans correlation coefficient. Receiver operating characteristic (ROC) curves were generated for AMH and AFC to compare ability of parameters to predict top quality or frozen embryos and the occurrence of a live birth. RESULTS Of the 137 women who had fresh embryo transfer, 52 became pregnant (32.1% pregnancy rate per cycle started) and 38 had a live birth (23.5% live birth rate per cycle started). Both AMH and AFC had highly significant correlations with the number of oocytes retrieved and the number of oocytes fertilized (P<0.001). The two markers were also significantly associated with the number of top quality embryos available for transfer and the number of embryos frozen (P<0.01). With regard to live birth, AMH performed better than AFC (P<0.01 and P<0.05, respectively), but both markers were more valuable in predicting the absence rather than the occurrence of live birth (negative predictive value 84%). CONCLUSIONS AMH and AFC are comparable predictors of oocytes retrieved and of the number of good quality embryos available for transfer and freezing. Prediction of live birth may help clinicians selecting patients suitable for single embryo transfer.