Ellen Anckaert

FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID–LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations

BACKGROUND Serum free thyroxine (FT4) testing in pregnancy is known to be challenging for immunoassays (IAs). We verified the reliability of FT4 results by 3 commercial IAs throughout pregnancy, by comparison of the pattern to that obtained with an equilibrium dialysis isotope dilution-mass spectrometry (ED ID-MS) candidate reference measurement procedure. METHODS Pregnant females (107) and age-matched non-pregnant controls (26) were enrolled. The IAs tested were those performed on the Cobas 6000 (Roche Diagnostics), ARCHITECT i2000SR (Abbott Diagnostics) and Immulite 2000 (Siemens Healthcare) platforms. RESULTS Compared to the controls (mean FT4: 18.2pmol/L), ED ID-MS gave in the late first trimester pregnancy an 8.8% lower (p<0.05) mean; in the second trimester it was 29.1% lower (p<0.001), to stabilize in the third trimester (p=0.99). Similar observations were made for the Cobas and Immulite IAs. The ARCHITECT IA showed no significant decrease in the late first trimester (mean 13.5pmol/L versus 13.6pmol/L in controls), but a significant, less pronounced, decrease in the second and third trimesters (15% and 14.4%, respectively). All IAs were susceptible towards alterations in T4 binding proteins during pregnancy. CONCLUSION We proved that IAs may give a FT4 pattern during pregnancy similar to that obtained by ED ID-MS.

Culture of oocytes and risk of imprinting defects

BACKROUND Follicle culture and oocyte in vitro maturation (IVM) are emerging assisted reproductive technologies with potentially important future applications in the fertility clinic. There is concern that these technologies might interfere at the epigenetic level and, in particular, with genomic imprinting. The timely acquisition of correct imprinting patterns in oocytes and the maintenance of genomic imprinting after fertilization are both required for normal embryonic development. METHODS A systematic literature search in Pubmed was performed and all publications reporting on the effects of follicle culture, IVM or ovarian tissue culture on genomic imprinting were retained. RESULTS Mouse ovarian tissue culture studies, mouse in vitro follicle culture studies and a single bovine IVM study generally showed correct imprinted DNA methylation establishment in oocytes. Influences of treatment and suboptimal culture conditions in mouse follicle culture indicate that imprinting establishment in oocytes is a robust process. This is in contrast with preimplantation embryo culture-induced epigenetic defects reported in mice. For human IVM, no definitive conclusion on imprinting establishment can be drawn as well-designed studies are currently not available. CONCLUSIONS Animal models provide reassuring data on imprinting establishment in cultured oocytes, but further studies should assess the effect of oocyte culture on imprinting maintenance. Optimized IVM procedures should be assessed in well-designed human studies. Finally, epigenetic analysis should be performed in children born from pregnancies after IVM to draw definitive conclusions on the epigenetic safety of human IVM.

The value of anti-Müllerian hormone measurement in the long GnRH agonist protocol: association with ovarian response and gonadotrophin-dose adjustments

BACKGROUND This study evaluated the predictive value of serum and follicular fluid (FF) concentrations of anti-Müllerian hormone (AMH) with respect to treatment outcome variables in an IVF cycle. METHODS A retrospective analysis was performed with data from 731 normogonadotrophic women undergoing controlled ovarian stimulation after stimulation with highly purified menotrophin (HP-hMG) or rFSH following a long GnRH agonist protocol. RESULTS In both treatment groups, the serum AMH concentration at the start of the stimulation was significantly (P < 0.001) positively correlated with the serum levels of estradiol (HP-hMG: r = 0.45; rFSH: r = 0.55), androstenedione (HP-hMG: r = 0.50; rFSH: 0.49) and total testosterone (HP-hMG: r = 0.40; rFSH: r = 0.36) at the end of the stimulation as well as the number of oocytes retrieved (HP-hMG: r = 0.48; rFSH: r = 0.62), the AMH concentration in FF (HP-hMG: r = 0.55; rFSH: 0.61) and the serum progesterone concentration (HP-hMG: r = 0.39; rFSH: r = 0.50) at oocyte retrieval. For both treatments, serum AMH at the start of the stimulation was a good predictor of the need to increase or decrease the gonadotrophin dose on stimulation day 6 and of ovarian response below (<7 oocytes) or above (>15 oocytes) the target. No significant relationships were observed between serum AMH and embryo quality or ongoing pregnancy. CONCLUSION The serum AMH concentration at the start of the stimulation in IVF patients down-regulated with GnRH agonist in the long protocol revealed a positive relationship with ovarian response to gonadotrophins in terms of oocytes retrieved and accompanying endocrine response. AMH is a good predictor of the need for gonadotrophin-dose adjustment on stimulation day 6 for patients with a fixed starting dose, but a poor predictor of embryo quality and pregnancy chances in individual patients.

Unaltered imprinting establishment of key imprinted genes in mouse oocytes after in vitro follicle culture under variable follicle-stimulating hormone exposure

Imprinted genes are differentially methylated during gametogenesis to allow parental-specific monoallelic expression of genes. During mouse oogenesis, DNA methylation at imprinted genes is established during the transition from primordial to antral follicle stages. Studies in human and mouse suggest aberrant imprinting in oocytes following in vitro maturation and after superovulation with high doses of gonadotrophines. The exact mechanisms leading to aberrant imprinting are unknown. We examined the methylation status of differentially methylated regions of key imprinted genes (by bisulphite sequencing) in mouse metaphase II oocytes, grown in a long-term pre-antral follicle culture system and matured in vitro, in the presence of a physiological (10 IU/L) and a high (100 IU/L) recombinant FSH dose. Our results showed a normal DNA methylation at the studied regulatory sequences of Snrpn, Igf2r and H19, demonstrating that 1) prolonged culture and in vitro maturation do not per se modify the establishment of imprinting in oocytes and 2) supraphysiological FSH doses do not induce aberrant DNA methylation at the studied regulatory sequences in this system.

Limited value of ovarian function markers following orthotopic transplantation of ovarian tissue after gonadotoxic treatment.

CONTEXT In young women, some treatments for cancer or other conditions (such as sickle cell anemia) may give rise to primary ovarian insufficiency. Ovarian transplantation is one of the available options for fertility preservation, with highly variable pregnancy rates. OBJECTIVE The objective of the study was to investigate markers of ovarian reserve and ovarian function in women up to 7 yr after orthotopic ovarian transplantation. Secondary objectives were to assess the relationship between markers of ovarian reserve and pregnancy rate along with the duration of ovarian function. DESIGN This was a prospective cohort study in 10 women, with a mean follow-up of 2.5 yr. SETTING The study was conducted at a university hospital in Brussels, Belgium. PATIENTS Patients included 10 women who were about to receive or had previously received gonadotoxic treatment. In seven women cryopreservation of ovarian tissue was performed before starting treatment. Subsequently autografts were orthotopically transplanted in these women. Three women, who had already developed primary ovarian insufficiency due to treatment, underwent orthotopic transplantation of ovarian allograft tissue originating from their human leukocyte antigen-compatible sisters. MAIN OUTCOME MEASURES Serum concentrations of FSH, LH, estradiol, inhibin B, and anti-Müllerian hormone (AMH) were measured. RESULTS On average, first menses took place after 4.7 months. Duration of graft functioning varied from 2 to more than 60 months. FSH concentrations remained elevated, whereas estradiol levels normalized and AMH was low to undetectable. Inhibin B varied among women and was not associated with the duration of ovarian function (hazard ratio 0.966, 95% confidence interval 0.881-1.059). Two spontaneous pregnancies occurred. Endocrine characteristics were not significantly different in these women. CONCLUSIONS Low AMH and inhibin B concentrations may suggest decreased ovarian reserve in women after ovarian transplantation. AMH and inhibin B levels may not be associated with the duration of ovarian graft function or probability to achieve a pregnancy.

Clinical and biologic evaluation of ovarian function in women treated by bone marrow transplantation for various indications during childhood or adolescence

OBJECTIVE To evaluate which factors determine premature ovarian failure after bone marrow transplantation (BMT) during childhood and adolescence. DESIGN Cross-sectional study. SETTING Academic teaching hospital. PATIENT(S) Thirty-five women with previous allogeneic (n = 19) or autologous (n = 16) BMT for benign (n = 12) or malignant disease (n = 23). Mean age at BMT was 9.8 ± 5.2 years. Eighteen patients had received total body irradiation (TBI). Twenty-three (66%) were premenarchal at the time of BMT. INTERVENTION(S) Evaluation of ovarian function. MAIN OUTCOME MEASURE(S) Retrospective analysis of gynecologic history and hormone measurements (FSH, E(2), and antimüllerian hormone [AMH]) in relation to initial pathology, treatment protocols, age, and menarchal status at the time of BMT and time elapsed since BMT. RESULT(S) Clinical evidence of persistent ovarian function after BMT was found in 46% of patients (16/35), but low AMH concentrations (<1.2 μg/L) were observed in 85% of patients, including a large subset (2/3) of clinically eugonadal subjects. Age ≤10 years at the time of BMT and absence of TBI were significantly and independently associated with higher rates of clinically proven persistent ovarian function at the time of evaluation. CONCLUSION(S) After BMT, ovarian function is impaired in the majority of women, even without clinical signs of premature ovarian failure. This impairment is mostly related to older age at the time of treatment and conditioning treatment with TBI.

Effects of Low Methyl Donor Levels in Culture Medium During Mouse Follicle Culture on Oocyte Imprinting Establishment

Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. We previously demonstrated establishment of normal imprinting at four key imprinted genes in mouse metaphase II oocytes after in vitro follicle culture. Commercially available culture media feature a wide range of methyl donor levels. The aim of the present study was to examine the effect of low methyl donor (methionine, vitamin B12, folic acid, choline, and vitamin B6) levels during follicle culture on acquisition of DNA methylation at imprinted genes in mouse oocytes. Follicle culture performed under low methyl donor levels led to decreased antral follicle development (mean [SD] antral follicle rate, 87.5% [12.6%] vs. 97.7% [4.3%] in control conditions; P < 0.05) and to a dramatic decrease in polar body (PB) oocyte rate (mean [SD] PB oocyte rate, 38.7% [25.5%] vs. 96.1% [7.1%]; P < 0.001). The methylation status of differentially methylated regions (DMRs) of four key imprinted genes was studied (by bisulphite sequencing) in normal-looking PB and germinal vesicle breakdown-arrested oocytes obtained from follicle culture under low methyl donor levels. DMRs of Snrpn, Igf2r, and H19 showed no alteration in DNA methylation, but at Mest DMR in PB oocytes, we found a significant reduction in DNA methylation compared to that in control follicle culture (DNA methylation, 89.9% and 98.2%, respectively; P = 0.0014). In conclusion, restriction of methyl donors during follicle culture led to a dramatic decrease in PB oocyte rate but induced no or only minor DNA methylation alterations at the studied regulatory sequences of key imprinted genes in oocytes.

Vitamin D deficiency and pregnancy rates in women undergoing single embryo, blastocyst stage, transfer (SET) for IVF/ICSI

STUDY QUESTION What is the influence of vitamin D deficiency on pregnancy rates among women undergoing IVF/ICSI and Day 5 (blastocyst stage) single embryo transfer (SET)? SUMMARY ANSWER Vitamin D deficiency results in significantly lower pregnancy rates in women undergoing single blastocyst transfer. WHAT IS KNOWN ALREADY Preliminary experiments have identified the presence of vitamin D receptors in the female reproductive system. However, results regarding the effect of vitamin D deficiency on clinical outcomes are conflicting. None of the previous studies adopted a SET strategy. STUDY DESIGN, SIZE, DURATION Serum vitamin D concentration was measured retrospectively in patients who underwent SET on Day 5. Overall 368 consecutive infertile women treated within a period of 15 months were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients underwent ovarian stimulation for IVF/ICSI and Day 5 SET. Serum samples were obtained 7 days prior to embryo transfer and stored frozen at -20°C. Samples were collectively analyzed for their 25-OH vitamin D content. Vitamin D deficiency was defined as serum 25-OH vitamin D levels <20 ng/ml in accordance with the Institute of Medicine and the Endocrine Society clinical practice guidelines. MAIN RESULTS AND THE ROLE OF CHANCE Clinical pregnancy rates were significantly lower in women with vitamin D deficiency compared with those with higher vitamin D values (41 versus 54%, P = 0.015).Logistic regression analysis was performed to identify whether vitamin D deficiency is independently associated with clinical pregnancy rates after controlling for 16 potential confounding factors. According to our results vitamin D deficiency was independently associated with lower clinical pregnancy rates, odds ratios [ORs (95% confidence interval (CI) 0.61 (0.39-0.95)] for vitamin D deficiency (deficient versus non-deficient women), P = 0.030. Finally, even when restricting our analysis to women undergoing elective SET (274 patients), vitamin D deficiency was again independently associated with pregnancy rates [OR (95% CI) 0.56 (0.33-0.93), P = 0.024]. LIMITATIONS, REASONS FOR CAUTION Our results refer only to patients undergoing Day 5 SET. Although vitamin D deficiency appears to compromise pregnancy rates in this population, no guidance can be provided regarding a potential relationship between vitamin D deficiency and ovarian reserve or response to ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS Vitamin D deficiency impairs pregnancy rates in women undergoing single blastocyst transfer. Future prospective confirmatory studies are needed to validate our results and examine the exact underlying mechanism by which vitamin D levels may impair pregnancy rates in infertile women undergoing IVF/ICSI. STUDY FUNDING/COMPETING INTERESTS None declared.

Prospective study into the value of the automated Elecsys antimüllerian hormone assay for the assessment of the ovarian growing follicle pool

OBJECTIVE To evaluate a new fully automated assay measuring antimüllerian hormone (AMH; Roche Elecsys) against antral follicle count in women of reproductive age. DESIGN Prospective cohort study. SETTING Hospital infertility clinics and academic centers. PATIENT(S) Four hundred fifty-one women aged 18 to 44 years, with regular menstrual cycles. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) AMH and antral follicle count (AFC) determined at a single visit on day 2-4 of the menstrual cycle. RESULT(S) There was a statistically significant variance in AFC but not in AMH between centers. Both AFC and AMH varied by age (overall Spearman rho -0.50 for AFC and -0.47 for AMH), but there was also significant between-center variation in the relationship between AFC and age but not for AMH. There was a strong positive correlation between AMH and AFC (overall spearman rho 0.68), which varied from 0.49 to 0.87 between centers. An agreement table using AFC cutoffs of 7 and 15 showed classification agreement in 63.2%, 56.9% and 74.5% of women for low, medium, and high groups, respectively. CONCLUSION(S) The novel fully automated Elecsys AMH assay shows good correlations with age and AFC in women of reproductive age, providing a reproducible measure of the growing follicle pool.

Human in vitro oocyte maturation is not associated with increased imprinting error rates at LIT1, SNRPN, PEG3 and GTL2

STUDY QUESTION Does in vitro maturation (IVM) of cumulus-enclosed germinal vesicle (GV) stage oocytes retrieved from small antral follicles in minimally stimulated cycles without an ovulatory hCG dose induce imprinting errors at LIT1, SNRPN, PEG3 and GTL2 in human oocytes? SUMMARY ANSWER There is no significant increase in imprinting mutations at LIT1, SNRPN, PEG3 and GTL2 after IVM of cumulus-enclosed GV oocytes from small antral follicles in minimally stimulated cycles without hCG priming. WHAT IS KNOWN ALREADY Animal models have generally demonstrated correct methylation imprint establishment for in vitro grown and matured oocytes. For human IVM, well-designed studies allowing conclusions on imprint establishment are currently not available. STUDY DESIGN, SIZE, DURATION Immature oocyte-cumulus complexes from 2 to 9 mm follicles were retrieved in polycystic ovary syndrome (PCOS) subjects in minimally stimulated cycles without hCG priming and matured in vitro. In vivo grown oocytes were retrieved after conventional ovarian stimulation for IVF/ICSI or after ovulation induction. Imprinting error rates at three maternally methylated (LIT1, SNRPN and PEG3) and one paternally methylated (GTL2) imprinted genes were compared in 71 in vitro and 38 in vivo matured oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS The limiting dilution bisulfite sequencing technique was applied, allowing increased sensitivity based on multiplex PCR for the imprinted genes and the inclusion of non-imprinted marker genes for cumulus cell DNA contamination. MAIN RESULTS AND THE ROLE OF CHANCE In vitro as well as in vivo matured oocytes showed only a few abnormal alleles, consistent with epimutations. The abnormalities were more frequent in immature than in mature oocytes for both groups, although no significant difference was reached. There was no statistically significant increase in imprinting errors in IVM oocytes. LIMITATIONS, REASONS FOR CAUTION This single cell methylation analysis was restricted to a number of well-selected imprinted genes. Genome-wide methylation analysis of single human oocytes is currently not possible. WIDER IMPLICATIONS OF THE FINDINGS IVM is a patient-friendly alternative to conventional ovarian stimulation in PCOS patients and is associated with reduced gonadotrophin costs and avoidance of OHSS. The results of this study show for the first time that optimized human IVM procedures have no significant effects on the establishment of maternal DNA methylation patterns at LIT1, SNRPN, PEG3 and GTL2. STUDY FUNDING/COMPETING INTERESTS This study was supported by research funds from Agentschap voor Innovatie door Wetenschap en Technologie (IWT-TBM 110680), Wetenschappelijk Fonds Willy Gepts (WFWG 2011) and German Research Foundation (HA 1374/12-2). There are no competing interests.