C. Ortega-Hrepich

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.

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.

Corifollitropin alfa followed by rFSH in a GnRH antagonist protocol for poor ovarian responder patients: an observational pilot study

OBJECTIVE To identify whether women with poor ovarian response may benefit from treatment with corifollitropin alfa in a GnRH antagonist protocol. DESIGN Retrospective pilot study. SETTING University-based tertiary care center. PATIENT(S) Poor ovarian responders fulfilling the Bologna criteria developed by European Society for Human Reproduction and Embryology Consensus Group. INTERVENTION(S) Corifollitropin alfa (150 μg) followed by 300 IU rFSH in a GnRH antagonist protocol. MAIN OUTCOME MEASURE(S) Endocrinologic profile and ongoing pregnancy rates. RESULT(S) Among 43 women treated with corifollitropin alfa, mean E(2) levels showed an increasing pattern during the follicular phase, reaching 825 ng/L on the day of hCG administration, whereas FSH values showed a marked increase during the first 5 days, reaching a mean value of 35 IU/L and remaining above 20 IU/L during the late follicular phase. Cycle cancellation rate was 32.6% and embryo transfer rate 53.3%. Five patients (11.7%) had a positive hCG test and three (7%) had an ongoing pregnancy. Ongoing pregnancy rates were 11.1% per oocyte retrieval and 13% per embryo transfer. Ongoing pregnancy rates per patient did not significantly differ compared with a cohort of patients treated during 2011 with the standard protocol for poor responders in our center (short agonist-hMG) (7% vs. 6.3%). CONCLUSION(S) Treatment of poor ovarian responders, as described by the Bologna criteria, with corifollitropin alfa in a GnRH antagonist protocol results in low pregnancy rates, similarly to conventional stimulation with a short agonist protocol.

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.

Addition of highly purified HMG after corifollitropin alfa in antagonist-treated poor ovarian responders: a pilot study

STUDY QUESTION Will sequential administration of highly purified (hp)-HMG after corifollitropin alfa in a GnRH antagonist protocol benefit women with poor ovarian response according to the Bologna criteria? SUMMARY ANSWER Corifollitropin alfa followed by hp-HMG in a GnRH antagonist protocol results in very promising pregnancy rates, albeit only in young (<40 years old) poor ovarian responders fulfilling the Bologna criteria. WHAT IS KNOWN ALREADY Poor ovarian responders fulfilling the Bologna criteria have a very poor prognosis in terms of successful IVF outcome. Although a recent study demonstrated low pregnancy rates in this group of patients after treatment with corifollitropin alfa followed by recombinant FSH in a GnRH antagonist protocol, previous studies showed that the addition of LH activity in 36- to 39-year-old women significantly increases implantation rates. STUDY DESIGN, SIZE, DURATION In this retrospective pilot study, we included poor ovarian responders fulfilling the Bologna criteria treated with a completely novel protocol, with corifollitropin alfa followed by hp-HMG in a GnRH antagonist setting. Overall, 51 patients were treated within a period of 1 year (August 2011-August 2012). PARTICIPANTS/MATERIALS, SETTING, METHODS Patients received 150 μg corifollitropin alfa on second day of the menstrual cycle followed by a fixed daily dose of 0.25 mg of GnRH antagonist on Day 7 of the cycle onwards. On the ninth day of the cycle, a daily fixed dose of 300 IU hp-HMG was administered until the day of ovulation triggering. The primary outcome was ongoing pregnancy rate per patient. MAIN RESULTS AND THE ROLE OF CHANCE Among 47 eligible women, 29 patients were <40 years old and 18 patients were ≥ 40 years old. No differences were observed in endocrine profile, number of cycles with oocyte retrieval (66 versus 67%) and cycles with embryo transfer (62 versus 61%) in women <40 versus ≥ 40 years old, respectively. However, 8 of the 29 women <40 years old had an ongoing pregnancy (28%) compared with 0 of 18 patients who were ≥ 40 years of age (P = 0.017). LIMITATIONS, REASONS FOR CAUTION Owing to the specific retrospective study design, bias cannot be ruled out and these results should not be extrapolated to other treatment protocols for poor ovarian responders. Therefore, caution should be taken when interpreting the results. WIDER IMPLICATIONS OF THE FINDINGS The promising results from this pilot study of corifollitropin alfa followed by hp-HMG stimulation indicate a potential beneficial effect in young poor ovarian responders fulfilling the Bologna criteria. The data provide the rationale for performing a randomized controlled trial to determine if there is sound evidence for a clinical introduction of this protocol. STUDY FUNDING/COMPETING INTEREST(S) No conflicts of interest to declare. No specific funding was received for this study.

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.

Effect of heparin exposure before in vitro maturation culture on oocyte maturation

OBJECTIVE: To assess the effect of heparin exposure to immature oocytes before IVM culture. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Since 2010 we have offered IVM treatment to patients with PCO/PCOS. Patients received 3 daily injections of 150 IUHP-HMG from cycle day 3. Embryology data and cumulative clinical outcomes were analyzed according towhether or not heparin was used in the collection media during IVM oocyte retrieval (OR). In 79 cycles OR was performed in a collection system without heparin (group1 -G1). In 53 cycles cumulus-oocyte complexes (COCs) were exposed for 30 minutes to heparin Leo (10IU/ml) that had been added to the collection media (group2 -G2).