L. Guzman

Clinical outcome of non–hCG-primed oocyte in vitro maturation treatment in patients with polycystic ovaries and polycystic ovary syndrome

OBJECTIVE To compare clinical outcomes of fresh embryo transfer (ET) and vitrified-warmed ET in an artificial endometrial priming cycle in patients with polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) who underwent oocyte in vitro maturation (IVM) in non-hCG-primed cycles. DESIGN Prospective cohort study. SETTING University-based tertiary referral center. PATIENT(S) Thirty-nine consecutive patients <37 years old with PCO or PCOS, who underwent 73 cycles of immature oocyte retrieval. INTERVENTION(S) Immature oocyte collection after ovarian stimulation with a cumulative dose of 450 IU uFSH or highly purified hMG, but without hCG priming. IVM of oocytes followed by ET if endometrium thickness ≥ 6 mm. Embryo vitrification at the cleavage stage. ET in an artificial cycle. MAIN OUTCOME MEASURE(S) Implantation rate (IR) and clinical pregnancy rate (CPR). RESULT(S) Fresh ET after IVM resulted in an IR of 6.9% (5/72) per ET and a CPR of 9.4% (5/53). ET of vitrified-warmed IVM embryos in an artificial cycle resulted in significantly better outcomes (IR 21.9% [7/32] and CPR 31.8% [7/22] per ET). CONCLUSION(S) A non-hCG-primed IVM system in PCO or PCOS performs poorly when embryos are transfered in a fresh cycle. Transfer of vitrified-warmed IVM embryos in an artificial cycle leads to significantly improved clinical outcomes. These data illustrate that IVM embryos in PCO or PCOS have good survival rates and suggest that hCG may be needed to support endometrial receptivity in the fresh IVM cycle.

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.

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence

STUDY QUESTION Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? SUMMARY ANSWER Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. WHAT IS KNOWN ALREADY In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. STUDY DESIGN, SIZE, DURATION An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on individual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. MAIN RESULTS AND THE ROLE OF CHANCE In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence; however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. LIMITATION, REASONS FOR CAUTION Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. WIDER IMPLICATIONS OF THE FINDINGS This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

Developmental capacity of in vitro–matured human oocytes retrieved from polycystic ovary syndrome ovaries containing no follicles larger than 6 mm

OBJECTIVE To test the developmental competence of oocytes in a nonhCG-triggered in vitro maturation (IVM) system when oocyte-cumulus complexes (OCC) are retrieved from antral follicles with a diameter of <6 mm. DESIGN Prospective cohort study. SETTING Tertiary university-based referral center. PATIENT(S) From January 2010 to September 2011, 121 patients with polycystic ovaries/polycystic ovary syndrome underwent 239 IVM cycles in total. In 58 of these cycles (44 patients), all antral follicles had a diameter of <6 mm on the day of oocyte retrieval. INTERVENTION(S) NonhCG-triggered IVM of oocytes, fresh or vitrified/warmed embryo transfer (ET). MAIN OUTCOME MEASURE(S) Oocyte diameter, maturation rate, fertilization rate, embryo development and morphology, implantation rate, clinical pregnancy rate, ongoing pregnancy rate. RESULT(S) Oocyte retrieval yielded 16.7 OCC/cycle, and 50.8% of oocytes completed IVM. The mean oocyte diameter increased from 108.8 ± 4.3 μm to 111.9 ± 4.1 μm after IVM. Mean fertilization rate was 63.7%, and 45.4% of 2-pronuclei oocytes developed into a morphologically good-quality embryo on day 3 after intracytoplasmic sperm injection. Fresh ET resulted in two ongoing pregnancies (2/37; 5.4%). Deferred vitrified-warmed ET led to an ongoing pregnancy rate of 34.6% (9/24). Three healthy babies were born and eight pregnancies were still ongoing. CONCLUSION(S) Oocytes retrieved from follicles with a diameter of <6 mm grow during a 40-hour IVM culture can acquire full competence in vitro, as illustrated by their development into healthy offspring. Endometrial quality appears to be a crucial determinant of pregnancy after nonhCG-triggered IVM.

Predictors of ovarian response in women treated with corifollitropin alfa for in vitro fertilization/ intracytoplasmic sperm injection

OBJECTIVE To identify predictors of ovarian response in women undergoing ovarian stimulation with corifollitropin alfa in a GnRH antagonist protocol and determine specific thresholds for the prediction of low and excessive responders. DESIGN Retrospective cohort study. SETTING University-based tertiary care center. PATIENT(S) Infertile women undergoing ovarian stimulation for in vitro fertilization/intracytoplasmic sperm injection. INTERVENTION(S) Controlled ovarian hyperstimulation with corifollitropin alfa in a GnRH antagonist protocol. MAIN OUTCOME MEASURE(S) Relationship between ovarian reserve tests and ovarian response. RESULT(S) Antimüllerian hormone (AMH) and antral follicle count (AFC) were the only independent predictors for low and excessive ovarian response. In prediction of excessive response, the area under the receiver operating characteristic curve [AUC (95% CI)] for AMH was 0.890 (0.832-0.947) and 0.897 (0.829-0.964) for AFC. The optimal thresholds for identifying excessive responders were 3.52 ng/mL for AMH (sensitivity 89.5, specificity 83.8) and 16 for AFC (sensitivity 80.0, specificity 84.5). AMH and AFC also predicted low ovarian response: AUCs AMH 0.836 (0.783-0.889) and AFC 0.830 (0.767-0.894). The optimal thresholds for predicting low response were 1.37 ng/mL for AMH (sensitivity 74.1, specificity 77.5) and 8 for AFC (sensitivity 72.2, specificity 84.6). For both excessive and low ovarian responses, a logistic regression model combining the biomarkers was associated with improved discrimination. CONCLUSION(S) AMH and AFC are the best predictors for low and excessive response in women treated with corifollitropin alfa in an antagonist protocol. Using AMH and AFC to select suitable candidates for treatment with corifollitropin alfa may result in a safe and convenient stimulation.

Human antral follicles <6 mm: a comparison between in vivo maturation and in vitro maturation in non-hCG primed cycles using cumulus cell gene expression

Within the context of an oocyte in vitro maturation (IVM) program for reproductive treatment, oocyte cumulus complexes (COCs) derived from follicles <6 mm in patients with PCOS were matured in vitro. Key transcripts related to meiotic maturation (FSHR, LHCGR, EGFR, PGR) and oocyte competence (AREG, ADAMTS, HAS2, PTGS2) were quantified in cumulus cells (CCs) before and after maturation. Control CC samples were collected from PCOS and normo-ovulatory patients who had undergone conventional gonadotrophin stimulation for IVF/ICSI. Additional control samples from a non-stimulated condition were obtained ex vivo from patients undergoing ovariectomy for fertility preservation. Expression data from CCs from follicles with a diameter of <6 mm before (IVM-CCs) and after in vitro maturation (IVM-CCs) were obtained after pooling CCs into four groups in relation to the percentage of matured (MII) oocytes obtained after 40 h of IVM (0; 40-60; 61-80; 100% MII) and values were compared with in vivo matured controls (IVO-CCs). Genes encoding key receptors mediating meiotic resumption are expressed in human antral follicles of <6 mm before and after IVM. The expression levels of FSHR, EGFR and PGR in CCs were significantly down-regulated in the IVO-CCs groups and in the 100% MII IVM group compared with the BM groups; all the receptors studied in the 100% MII IVM group reached an expression profile similar to that of IVO-CCs. However, after maturation in a conventional IVF/ICSI cycle, IVO-CCs from large follicles contained significantly increased levels of ADAMTS1, AREG, HAS2 and PTGS2 compared with IVM-CCs and IVM-CCs; the expression patterns for these genes in all IVM-CCs were unchanged compared with IVM-CCs. In conclusion, genes encoding receptors involved in oocyte meiotic resumption appeared to be expressed in CCs of small human antral follicles. Expression levels of genes-encoding factors reflecting oocyte competence were significantly altered in IVM-CCs compared with in vivo matured oocytes from large follicles. Observed differences might be explained by the different stimulation protocols, doses of gonadotrophin or by the intrinsic differences between in vivo and in vitro maturation.

A prediction model to select PCOS patients suitable for IVM treatment based on anti-Müllerian hormone and antral follicle count

STUDY QUESTION Which baseline patient characteristics can help assisted reproductive technology practitioners to identify patients who are suitable for in-vitro maturation (IVM) treatment? SUMMARY ANSWER In patients with polycystic ovary syndrome (PCOS) who undergo oocyte IVM in a non-hCG-triggered system, circulating anti-Müllerian hormone (AMH), antral follicle count (AFC) and total testosterone are independently related to the number of immature oocytes and hold promise as outcome predictors to guide the patient selection process for IVM. WHAT IS ALREADY KNOWN Patient selection criteria for IVM treatment have been described in normo-ovulatory patients, although patients with PCOS constitute the major target population for IVM. With this study, we assessed the independent predictive value of clinical and endocrine parameters that are related to oocyte yield in patients with PCOS undergoing IVM. STUDY DESIGN, SIZE, DURATION Cohort study involving 124 consecutive patients with PCOS undergoing IVM whose data were prospectively collected. Enrolment took place between January 2010 and January 2012. Only data relating to the first IVM cycle of each patient were included. PARTICIPANTS/MATERIALS, SETTING, METHOD Patients with PCOS underwent oocyte retrieval for IVM after minimal gonadotrophin stimulation and no hCG trigger. Correlation coefficients were calculated to investigate which parameters are related to immature oocyte yield (patients age, BMI, baseline hormonal profile and AMH, AFC). The independence of predictive parameters was tested using multivariate linear regression analysis. Finally, multivariate receiver operating characteristic (ROC) analyses for cumulus oocyte complexes (COC) yield were performed to assess the efficiency of the prediction model to select suitable candidates for IVM. MAIN RESULTS AND THE ROLE OF CHANCE Using multivariate regression analysis, circulating baseline AMH, AFC and baseline total testosterone serum concentration were incorporated into a model to predict the number of COC retrieved in an IVM cycle, with unstandardized coefficients [95% confidence interval (CI)] of 0.03 (0.02-0.03) (P < 0.001), 0.012 (0.008-0.017) (P < 0.001) and 0.37 (0.18-0.57) (P < 0.001), respectively. Logistic regression analysis shows that a prediction model based on AMH and AFC, with unstandardized coefficients (95% CI) of 0.148 (0.03-0.25) (P < 0.001) and 0.034 (-0.003-0.07) (P = 0.025), respectively, is a useful patient selection tool to predict the probability to yield at least eight COCs for IVM in patients with PCOS. In this population, patients with at least eight COC available for IVM have a statistically higher number of embryos of good morphological quality (2.9 ± 2.3; 0.9 ± 0.9; P < 0.001) and cumulative ongoing pregnancy rate [30.4% (24 out of 79); 11% (5 out of 45); P = 0.01] when compared with patients with less than eight COC. ROC curve analysis showed that this prediction model has an area under the curve of 0.7864 (95% CI = 0.6997-0.8732) for the prediction of oocyte yield in IVM. LIMITATIONS, REASONS FOR CAUTION The proposed model has been constructed based on a genuine IVM system, i.e. no hCG trigger was given and none of the oocytes matured in vivo. However, other variables, such as needle type, aspiration technique and whether or not hCG-triggering is used, should be considered as confounding factors. The results of this study have to be confirmed using a second independent validation sample. WIDER IMPLICATIONS OF THE FINDINGS The proposed model could be applied to patients with PCOS after confirmation through a further validation study. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a research grant by the Institute for the Promotion of Innovation by Science and Technology in Flanders, Project number IWT 070719.

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium

STUDY QUESTION Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? SUMMARY ANSWER The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. WHAT IS KNOWN ALREADY Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. STUDY DESIGN, SIZE, DURATION Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). PARTICIPANTS/MATERIALS, SETTING, METHODS Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. MAIN RESULTS AND THE ROLE OF CHANCE Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. LIMITATIONS, REASONS FOR CAUTION Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. STUDY FUNDING/COMPETING INTERESTS This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.

The effect of ovarian puncture on the endocrine profile of PCOS patients who undergo IVM.

BackgroundTo examine whether ovarian puncture for immature oocyte retrieval and in-vitro maturation (IVM) has an effect on the endocrine profile of patients with polycystic ovary syndrome (PCOS).MethodsTwenty-two consecutive patients with PCOS undergoing IVM treatment were included. Serum anti-Müllerian hormone (AMH), sex hormone-binding globulin (SHBG), total testosterone (TT) and luteinized hormone (LH) levels were analyzed at the start of the cycle, on the day of immature oocyte retrieval (OR) and at fixed intervals thereafter, for up to three months after OR.ResultsFive days after OR circulating AMH, TT, calculated free testosterone (FTc), and LH levels were significantly reduced and circulating SHBG was significantly increased. Two weeks after OR, TT, FTc and LH remained reduced, whereas circulating AMH and SHBG levels recovered to pre-puncture values. Three months after OR, all circulating hormone levels had recovered to baseline values.ConclusionOvarian puncture for the retrieval of immature oocytes and IVM in patients with PCOS has a significant impact on the ovarian endocrine profile, but this impact is brief and transient.

Causes and estimated incidences of sex-chromosome misdiagnosis in preimplantation genetic diagnosis of aneuploidy.

Preimplantation genetic diagnosis of aneuploidy (PGD-A) with comprehensive chromosome analysis has been known to improve pregnancy outcomes. Accuracy in detecting sex chromosomes becomes important when selecting against embryos at risk for sex-linked disorders. A total of 21,356 PGD-A cycles consisting of day-3 (cleavage) or day-5 (blastocyst) biopsies were received at the same laboratory for PGD-A via fluorescence in situ hybridization (FISH) or array comparative genome hybridization (aCGH) from multiple fertility centres. The misdiagnosis rates were 0.12% (Wilson 95% CI 0.05 to 0.25%) in day-3 FISH cycles, 0.48% (Wilson 95% CI 0.19 to 1.22%) in day-3 aCGH cycles and 0.0% (Wilson 95% CI 0 to 0.26) in day-5 aCGH cycles. Although rare, the likely causative biological event for true misdiagnosis is embryonic XX/XY mosaicism. Reanalysis of 1219 abnormal cleavage-stage research embryos revealed a 73% incidence of minor and major mosaicism. Only four (0.3%) embryos were found to be diploid and contained XX and XY cells that could potentially account for the misdiagnosis of sex. Our investigation identified errors leading to misdiagnosis and their attribution to specific events during PGD-A testing. The reported misdiagnosis rates suggest that PGD-A for sex determination is highly accurate, particularly when using aCGH applied to blastocyst biopsies.