Cheryl T. Fitzgerald

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.

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.

The optimal length of 'coasting protocol' in women at risk of ovarian hyperstimulation syndrome undergoing in vitro fertilization

Ovarian hyperstimulation syndrome (OHSS) is a serious and potentially life-threatening complication following ovarian stimulation for in vitro fertilization (IVF). Coasting is the practice whereby the gonadotrophins are withheld and the administration of human chorionic gonadotrophin (hCG) is delayed until serum oestradiol (E2) has decreased to what is considered to be a safe level, to prevent the onset of OHSS. This study aimed to assess the length of coasting on the reproductive outcome in women at risk of developing OHSS. Coasting was undertaken when the serum E2 concentrations were ≥17000 pmol/L but <21000 pmol/L. Daily E2 measurements were performed and hCG was administered when hormone levels decreased to <17000 pmol/L. Eighty-one women who had their stimulation cycles coasted were grouped according to the number of coasting days. Severe OHSS occurred in one case, which represented 1.2% of patients who underwent coasting because of an increased risk of developing the syndrome. No difference was found between cycles coasted for 1 – 3 days and cycles coasted for ≥4 days in terms of oocyte maturity, fertilization and embryo cleavage rates. Women in whom coasting lasted for ≥4 days had significantly fewer oocytes retrieved (P < 0.05) and decreased implantation rate (P < 0.05) compared to those coasted for 1 – 3 days. Pregnancy rate/embryo transfer and live birth rate did not differ between groups. In conclusion, coasting appears to decrease the risk of OHSS without compromising the IVF cycle pregnancy outcome. Prolonged coasting is, however, associated with reduced implantation rates, perhaps due to the deleterious effects on the endometrium rather than the oocytes.

Modelling the impact of single embryo transfer in a national health service IVF programme

BACKGROUND Greater use of single embryo transfer (SET) to reduce twin rates associated with IVF requires good information on prognostic factors and appropriate models of treatment outcomes. METHODS Using data from a cohort of 1198 IVF cycles, we have developed a statistical model of live birth and twin outcomes in terms of routinely measured clinical parameters. From this model, we predict potential outcomes if those who had two embryos transferred had actually received SET. RESULTS Embryo quality, age, FSH level, idiopathic diagnosis, sperm count, smoking and alcohol consumption are all significant factors predicting outcome. Couples with good embryos and good prognosis have a much greater risk of producing twins. In this cohort, to achieve a 10% twin rate would require 55% SET which, without selection of appropriate cycles, would lead to a reduction in success rate from ca. 21% to 17%. Selecting on the basis of twin risk can partially mitigate this reduction to give a success rate of 18.5%. CONCLUSIONS The use of SET to reduce twin rates will lead to a significant reduction in treatment success. Around half this reduction could be mitigated with careful selection of patients and cycles, including embryo quality.

Effect of age on the response of the hypothalamo-pituitary-ovarian axis to a combined oral contraceptive

OBJECTIVE To examine the effect of age on the response to treatment with a combined oral contraceptive. DESIGN Prospective, controlled clinical study. SETTING Reproductive medicine unit in a tertiary care university medical center. PATIENT(S) Twenty-six healthy female volunteers aged 21-45 years. INTERVENTION(S) After a control cycle, all the women were given a combined oral contraceptive containing 20 microg of ethinylestradiol with 75 microg of gestodene for three cycles. The women were examined through the posttreatment cycle. MAIN OUTCOME MEASURE(S) Pituitary and ovarian activity was assessed with ultrasound and measurement of ovarian steroids. RESULT(S) Follicular activity was observed in all treatment cycles, although ovulation was inhibited. Ovarian suppression was maximal in cycle 1. Mean endogenous E2 levels were lower during cycles 2 and 3 in the older group. Serum FSH levels were higher in the control cycle and on day 28 of the treatment cycles in the older group. Most women ovulated during the posttreatment cycle. CONCLUSION(S) Combined oral contraceptives did not inhibit all ovarian activity; maximal suppression was seen in cycle 1. Less follicular activity was observed in cycles 2 and 3 in the older group. Raised FSH levels with age reflect increasing ovarian resistance to follicular development.

Who needs ICSI? A nationwide UK survey on ICSI use

Background: Intracytoplasmic sperm injection (ICSI) has been a milestone in the treatment of male factor infertility. However ICSI is more expensive, demands more expertise, and involves more risk than conventional in vitro fertilisation (IVF). Currently there are large nationwide differences in ICSI usage, with some centres using ICSI for 21% of their IVF cycles and others for more than 80%. This is, most likely, due to differences in ICSI selection criteria but there are limited data on the criteria used. We have therefore carried out a national survey in the UK, the first, as far as we are aware, to examine different criteria used and their effect on ICSI usage and treatment outcomes. Methods: Centres which offer ICSI were identified using the Human Fertilisation and Embryology Authority (HFEA) website. Questionnaires were then posted to all centres which offer the procedure. Each centre received the questionnaire twice; the first was sent to the HFEA person responsible and a month later, a follow-up questionnaire was sent to the centre’s lead embryologist. Data were also extracted from the HFEA website. Results: 71 centres were identified and questionnaires returned from 43 (61%). When deciding to use ICSI, 43 (100%) of centres used sperm count, 93% used sperm motility, 76% used sperm morphology and 72% used anti-sperm antibodies. All centres stated that they would offer ICSI after failed fertilisation with conventional IVF and 38% of centres offered ICSI on patient request. No centres reported using other criteria for selection. The absolute values chosen for each criterion varied hugely between centres. Compared with the 2010 World Health Organization (WHO) guidelines of normal semen analyses, 32% of centres used a higher count, 50% a higher motility and 59% a higher morphology. Based on the WHO criteria, 27% of centres would use ICSI for sperm that were normal by all WHO criteria. Between centres, no significant difference in ICSI fertilisation rates was found. However, there was a significant negative correlation between increased ICSI usage and fertilisation rates by conventional IVF (p = 0.0058). Data obtained from the HFEA website failed to demonstrate an increase in live birth rate in centres using ICSI more frequently. Conclusion: ICSI usage varied widely, due to large differences in the ICSI selection criteria used, with many centres using ICSI for patients with normal semen parameters. Centres which used more ICSI did not report higher live birth rates. No evidence was found to suggest that higher ICSI usage increased overall fertilisation rates. These findings highlight the need for guidelines on when to use ICSI.

Sperm concentration and forward motility are not correlated with age in adolescents with an idiopathic varicocele and symmetrical testicular volumes.

AIM Idiopathic varicocele is a common condition that may impair fertility. Its treatment in children and adolescents is reserved for those patients who develop symptoms or testicular growth arrest. We evaluated the trends in sperm parameters among adolescent varicocele patients with symmetrical testicular volumes who have not undergone varicocelectomy. METHOD Data were prospectively collected from a single institution (2009 to 2014). Post-pubertal patients aged 12 to 17years produced semen samples by masturbation. Outcomes measured were semen volume, sperm concentration, and forward motility. Additional variables recorded included: a) testicular volume (ultrasound measurement), b) clinical varicocele grade, c) venous Doppler grading. Linear regression analysis was performed using Fishers transformation. P<0.05 was considered significant, and data are presented as median (IQ range). RESULTS Forty-one patients with a median age of 15.4 (15.0-15.9) years each provided a sperm sample during the study period. Thirty-five had grade 3 (visible) varicocele, and 6 had grade 2 (palpable) varicocele. All patients had spontaneous venous reflux on Doppler ultrasound, and none had undergone varicocelectomy prior to producing the sperm sample. Table 1 summarizes the sperm parameters according to patient age. The overall median sperm concentration was 37 (16-64) millions/ml and was not correlated with age. The overall median forward motility was 55% (44-64) and was not correlated with age. Thirty-four patients had normal sperm parameters, which remained within the WHO range of normality. CONCLUSIONS Following the European Association of Urology guidelines does not cause progressive deterioration of sperm parameters between the age of 12 and 17years.

Double ovarian stimulation (DuoStim) protocol for fertility preservation in female oncology patients

Abstract This article describes a revised ovarian stimulation protocol (DuoStim) for fertility preservation in female oncology patients which aims to maximise the number of gametes obtained with subsequent improvement in cumulative birth rate, without delaying cancer treatment. Ten patients diagnosed with malignancy between September 2014 and October 2015 were included. The patients were treated with the DuoStim protocol, undergoing two consecutive ovarian stimulation cycles and two oocyte retrievals. The primary outcome was the number of oocytes collected and vitrified during each oocyte retrieval and in total. The protocol was evaluated regarding medical risk and patients’ feedback. During the first oocyte collection 81 oocytes (61 metaphase II) were retrieved (mean = 8.1; range = 1–13) and during the second oocyte collection 82 oocytes (67 metaphase II) were retrieved (mean= 8.2; range = 1–19). A total of 163 oocytes (128 metaphase II) were collected (mean = 16.3; range = 6–32) and cancer treatment was not delayed for any of these patients. There were no cases of ovarian hyperstimulation syndrome recorded. More patients and long-term follow-up is needed to assess the efficacy and safety of the DuoStim protocol. However, these early results are encouraging, demonstrating an increase in number of mature oocytes retrieved during ovarian stimulation for oncology patients, without delaying cancer treatment.

A nationwide UK survey of female fertility preservation prior to cancer treatment

Abstract This survey examined the provision of fertility preservation for female oncology patients prior to cancer treatments, given their well-established gonadotoxic effects. Questionnaires were sent to all assisted conception units in the UK enquiring about the provision of oocyte or embryo cryopreservation, as well as funding for female oncology patients. In addition, data were obtained from the Human Fertilisation and Embryology Authority (HFEA) on the number of cryopreservation cycles in 2013–2014. Of the 60 responding units, 53 (88%) offered fertility preservation. However, only 6 (11%) units performed more than 25 oocyte or embryo cryopreservation cycles per year, with 33 units (62%) treating fewer than 10 women per year. A total of 44 (90%) reported some National Health Service (NHS) funding, but only 12 (23%) had funding granted automatically and only 26 (49%) could offer NHS funded treatment exempt from their local eligibility criteria for in vitro fertilisation (IVF). The HFEA data reported 154 NHS funded oocyte cryopreservation cycles in 2014. We conclude that the provision of fertility preservation is lacking and improvements can be made in the number of referrals from oncology, the provision of cryopreservation and the provision of NHS funding. Developing a national fertility preservation network and close liaison with oncology and Clinical Commissioning Groups are recommended.