In Vitro Maturation Treatment Research Articles

P-618 Impact of ovarian stimulation on outcome of oocyte in-vitro maturation (IVM) in women with polycystic ovaries: corifollitropin alfa (CFA) versus follitropin beta (FSH-B)

Abstract Study question Do corifollitropin alfa and follitropin beta have different effects on oocyte yield and live birth rates after IVM in women with polycystic ovaries? Summary answer In patients who underwent IVM, one injection of CFA resulted in lower oocyte retrieval rates, but similar birth rates compared to three injections of FSH-B. What is known already IVM involves the maturation of cumulus oocyte complexes (COCs) from antral follicles and has been offered to women with polycystic ovaries as an alternative for conventional ovarian stimulation (OS). Exogenous FSH is typically administered for two to five days in IVM cycles to enhance meiotic and developmental competence of immature oocytes in vivo. Previous studies have shown that the number of COCs is associated with ongoing pregnancy after IVM. Because one injection of CFA yields more oocytes compared to daily FSH-B injections in conventional OS protocols, CFA has the potential to combine patient-friendliness and maximised COC yield in IVM cycles. Study design, size, duration We conducted a randomised controlled superiority trial from 12/2017 to 12/2023. The primary endpoint was the number of COCs at collection. We randomised 145 patients to either one CFA 100μg injection or three daily injections of FSH-B 150IU. Laboratory and safety parameters, and pregnancy outcomes after frozen embryo transfer (FET) were analysed on an intention-to-treat basis. All cycles were scheduled using oral contraceptive pretreatment. Participants/materials, setting, methods Eligible patients were <37 years, had ≥24 antral follicles, and BMI 18-30 kg/m2. We analysed serum estradiol, progesterone, LH, and FSH on stimulation day 1 and 3, at oocyte retrieval (OR), and at OR + 6d. Oocyte retrieval (OR) was performed five days after the start of OS. No ovulation trigger was given. Monophasic IVM for 30h, ICSI and elective freeze-all were done in a tertiary fertility clinic. Data were analysed using STATA 13.0. Main results and the role of chance After randomisation, 70 patients underwent OR after FSH-B and 72 had OR after CFA. Hormone levels at OR were significantly different between FSH-B-treated (FSH 6.8 ± 2.8IU/L, LH 3.3 ± 2.7IU/L, E2 106.7 ± 147.0ng/L, Prog 0.19 ± 0.16μg/L) and CFA-treated patients (FSH 24.3 ± 7.9IU/L, LH 1.8 ± 1.6IU/L, E2 575.6 ± 516.2ng/L, Prog 0.29 ± 0.21 μg/L, p ≤ 0.001). On average, 43.1 ± 21.7(FSH-B) vs. 50.8 ± 29.2(CFA) follicles, all <10mm, were punctured during OR (p = 0.06). More COCs per follicle were retrieved after FSH-B (68.3 ± 31.5% vs. 51.0 ± 24.7%, p = 0.001), resulting in a tendency towards more COCs after FSH-B (31.4 ± 23.3), compared to CFA (23.4 ± 11.7, p = 0.07). Maturation rates after IVM were similar (49 ± 19% vs. 50 ± 15%, p = 0.79). In spite of a tendency towards more mature oocytes after FSH-B (16.0 ± 14.6 vs. 11.7 ± 7.9, p = 0.09), the number of good-quality cryopreserved embryos was similar (3.9 ± 2.9(FSH-B) vs. 3.5 ± 2.7(CFA), p = 0.36). Live birth rate (LBR) after the first FET (25.7%(FSH-B) vs. 34.7%(CFA), p = 0.24) and cumulative LBR six months after OR (40.0%(FSH-B) vs. 45.8%(CFA), p = 0.48) were comparable. None of the patients developed ovarian hyperstimulation syndrome (OHSS). Limitations, reasons for caution Outcomes are only applicable for patients with high antral follicle count (AFC) who are treated using a monophasic IVM culture system. The sample size was too small to draw significant conclusions for live birth rate. Wider implications of the findings In subfertile patients with high AFC who have IVM treatment, follicle priming with one injection of CFA is safe, convenient, and as efficacious as priming with FSH-B. The observation of lower oocyte retrieval rates after CFA compared to FSH-B in the specific setting of an IVM cycle merits further scrutiny. Trial registration number EudraCT 2017-002571-25

Clinical procedures for in vitro maturation treatment

To demonstrate clinical techniques for invitro maturation (IVM) treatment, including stimulation recommendations, small follicle pick-up procedures, and compact cumulus-oocyte complex (COC) search practice. This video utilizes live-action footage from surgery and embryology practice for a representative IVM treatment cycle, with step-by-step instructions and recommendations for practice procedures. Invitro fertilization (IVF) clinic. Patients undergoing IVM treatment. The patient(s) included in this video gave consent for publication of the video and posting of the video online, including on social media, the journal website, scientific literature websites, and other applicable sites. Identification of treatment cohorts, IVM definitions, and recommendations for stimulation treatments. A visual demonstration of COC extraction techniques from small antral follicles includes tubing, needle types, considerations when using double lumen or sheath needles, needle pressure, ultrasound, needle flushing, and aspiration technique. Visual demonstration of oocyte search and IVM preparation, including filtering follicular aspirate, prevention of COC cooling, identification of compact COCs, and general parameters of different IVM approaches. Clinical techniques for small follicle ovum pick up and compact COC identification for IVM treatment. Successful IVM treatment of patients can be achieved using minimal ovarian stimulation, effective small follicle retrieval, and efficient compact COC identification with flexibility in approach depending on clinical constraints and preference. Invitro maturation treatment is an efficacious and safe treatment for high ovarian reserve and hyper-responding patients undergoing IVF treatment, in which the retrieval of multiple immature COCs and their exvivo maturation can be achieved with little to no invivo stimulation. Practice procedures vary between treatment centers and IVM techniques. This video provides practice recommendations paired with a visual demonstration of techniques to assist in standardizing the approach and expanding the practice to more centers.

Outcomes of the Next In Vitro Fertilization Cycle in Women with Polycystic Ovary Syndrome after a Failed In Vitro Maturation Attempt.

In vitro maturation (IVM) is indicated in women with polycystic ovary syndrome (PCOS) who have a very good ovarian response during in vitro fertilization (IVF) and are therefore at high risk of ovarian hyperstimulation syndrome (OHSS). According to the latest practice committee document, IVM could be a major advance in assisted reproductive technology (ART) procedures (reduced cost and simplified treatment); nevertheless, retrospective studies of IVM versus IVF still demonstrate lower chances of a live birth with IVM. Could IVM prove to be an optimal first-line treatment approach? And limited information is available concerning the success of the subsequent IVF cycle after the failure of an IVM cycle. Does IVM treatment adversely affect the subsequent IVF cycle, and is this worth considering before performing the IVF cycle for women with PCOS? This prospective nested case-control study at the Peking University Reproductive Medicine center in China was performed between March 2018 and September 2020. Women aged 20-38 years with PCOS and infertility and who were scheduled for their first IVF attempt were eligible. A total of 351 women were randomly allocated to receive one cycle of unstimulated natural IVM (n = 175) or one cycle of standard IVF with a flexible GnRH antagonist protocol followed by hCG as an ovulation trigger (n = 176). This study involved 234 women (58 women with no blastocysts in the first IVM cycle and 158 women who underwent the first IVF cycle). Cumulative live birth rate at 12 months after oocyte retrieval and OHSS of a standard controlled ovarian stimulation (COS) IVF cycle were compared between 58 women in an IVF cycle following a failed IVM cycle and 158 women who underwent the first IVF cycle. No significant differences were found in the cumulative live birth rate (CLBR), ongoing pregnancy rate, or clinical pregnancy rate at 12 months after oocyte retrieval between the two groups (56.9% vs. 58.9%, p = 0.795; 58.6% vs. 60.8%, p = 0.776; and 84.5% vs. 76.0%, p = 0.178). The incidence of moderate-to-severe OHSS was not significantly different between the groups (6.9% vs. 5.7%, p = 0.742). Additionally, there were no significant differences in the total gonadotropin dose, stimulation duration, number of retrieved oocytes, number of retrieved mature oocytes, or fertilization rates. Even if the first IVM attempt failed in subfertile women with PCOS, comparable cumulative live birth rates were observed in the subsequent IVF cycle. IVM treatment does not adversely affect the subsequent IVF cycle.

P-510 Impact of ART on quality of life in predicted hyper-responders: conventional IVF versus in-vitro maturation of oocytes

Abstract Study question Do in-vitro maturation (IVM) of oocytes and conventional IVF (cIVF) have different effects on quality of life (QoL) in women with polycystic ovaries (PCO)? Summary answer Women with PCO who have IVM treatment experienced a lower impact on QoL compared to their counterparts who underwent cIVF. What is known already While studies in predicted hyper-responders have shown that success rates of IVM are lower compared to cIVF, cIVF is associated with more hormonal side effects and complications including OHSS. According to a recent discrete choice experiment among hyper-responders in the Netherlands, not only success rates may play a role in these women’s preferences for fertility treatment, but also projected risks, burden, and costs. It is currently unknown whether the increased efficiency of cIVF in hyper-responders may come at the expense of impact on QoL and whether IVM may be associated with a lower impact on mental health parameters. Study design, size, duration This is a single-centre, observational prospective study including 149 women with polycystic ovaries on ultrasound scan who had their first cycle of IVM (n = 75) or cIVF (n = 74) in a tertiary referral hospital. Patients were included between May 2017 and March 2021. Participants/materials, setting, methods Patients <37 years embarking on ART with their partner were asked to complete the Hospital Anxiety and Depression Scale (HADS) and the Fertility Quality of Life Questionnaire (FertiQoL) at three timepoints: intake at the fertility clinic (T1), at oocyte retrieval (T2) and after the first cycle outcome was known (T3). The primary objective was to determine the impact of ART on QoL. Statistical analysis included descriptive statistics and the use of general linear models. Main results and the role of chance In total, 124/149 (83.2%) patients completed questionnaires at two timepoints and 97/149 (65.1%) patients returned complete questionnaires. Mean age (28.92 ± 3.48y vs. 30.63 ± 3.23y, p = 0.003) was different in both groups. BMI (25.76 ± 5.81kg/m2 vs. 24.25 ± 4.83kg/m2, p = 0.089) and duration of infertility (27.12 ± 18.31 months vs. 31.00 ± 20.14 months, p = 0.270) were similar in IVM and cIVF patients, respectively. The distribution of PCOS phenotypes A, B, C and D, and PCOM was comparable in both groups (p = 0.142). Univariable analysis showed that women undergoing cIVF had worse side effects scores at T2 than women who had IVM (5.09 ± 3.24 vs. 3.08 ± 2.43, p < 0.001). According to multivariable ANOVA, the impact of IVM on anxiety and depression scores was similar to that of cIVF. Patients undergoing IVM had better scores for the FertiQol Subscale for Treatment Tolerability (68.54 ± 16.75 vs. 59.09 ± 22.68, p = 0.011), suggesting that cIVF caused more mental symptoms and daily life disruption. The Relational Subscale at T3 indicated that IVM patients suffered less impairment of sexuality and communication. Finally, social interactions at T3 were more severely affected by cIVF than IVM based on the social FertiQoL subscale (74.57 ± 19.64 vs 66.96 ± 17.60, p = 0.039). Limitations, reasons for caution The type of ART was not assigned randomly, thus selection bias was highly likely because of the study design. The willingness to trade off chance of pregnancy for lower burden and risks may have influenced the choice of ART type and may have modulated susceptibility to impact on QoL. Wider implications of the findings Patients utilizing IVM may expect fewer side effects, more tolerability to treatment and less impact on their relationship and social life compared to those who opt for cIVF. Lower efficiency may be an acceptable trade-off for the benefits of IVM. These findings should be corroborated by an RCT. Trial registration number clinical trials.gov NCT03066349

Characterization of the effects of heat stress on autophagy induction in the pig oocyte

BackgroundHeat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components.MethodsTo characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS).ResultsTo determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation.ConclusionsOur results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.

130 Expression of selected biomarker candidate genes to confer invitro maturation in Indian buffaloes

Invitro maturation (IVM) of oocytes is a crucial step and is directly related to better embryo production in buffaloes. Therefore, we planned to study gene expression of GDF9, HAS2, SPRY1, ARHGAP22, COL18A1, and GPC4 genes in IVM and immature cumulus–oocyte complexes (COCs). The COCs were recovered from follicles of slaughter origin ovaries of native buffaloes. COCs were observed under stereo zoom microscope and categorized in four grades according to morphology. Of the four grades, the first three grade COCs were considered and randomly allotted in two groups: immature treatment group (n=263) and IVM treatment group (n=272). IVM of COCs was carried out in 100-μL drops of BO-IVM medium overlaying embryo tested oil in a 35-mm petri dish under 5% CO2 in a 39.0°C incubator for 24h. Cumulus of COCs of both groups were removed by treating with 0.25% trypsin, and oocytes were stored in RNALater for future use. The expression of genes was evaluated using quantitative PCR, and the relative expression of each gene was calculated using the ΔΔCt method with efficiency correction. The logarithmic transformation of fold change (log2FC) of each candidate gene in the IVM oocyte group was computed against the immature oocyte group based on the observed cycle threshold values. Appropriate standard deviations were determined based on the observed deviations among the triplicates. The expression in the IVM treatment group of previously reported upregulated genes (GDF9, HAS2, SPRY1) was higher (up to 10-fold) compared with the immature treatment group (reference group). In the present study, relatively lower expression was observed for the other candidate genes (ARHGAP22, COL18A1, GPC4) in the bovine transcripts of oocyte, which were previously also reported as being downregulated.

Strategies of Infertility Treatment with Human Immature Oocytes

Human immature oocytes can be matured in vitro following culture. In vitro maturation (IVM) refers to maturation in culture of immature oocytes at different stages that may or may not have been exposed to short courses of gonadotropins. The source of immature oocytes is an important feature for the subsequent embryonic development and pregnancy, as well healthy live births. IVM is an efficient treatment that has already achieved significant outcomes in terms of acceptable pregnancies and implantation rates and resulted in the births of several thousands of healthy babies. As the development of IVM treatment continues, an attractive possibility for improving the already successful outcome is to combine a natural cycle in vitro fertilization (IVF) treatment with an immature oocyte retrieval followed by IVM of those immature oocytes. If the treatment processes can be simplified with immature oocyte retrieval, different types of infertile women may be able to take advantage of these treatments. Although IVM treatment is still considered experimental by the society, it is time to reconsider the IVM technological evolution. Mild stimulation IVF combined with IVM treatment represents a viable alternative to the standard treatment, and as data accumulate over time, it may prove to be an optimal first-line treatment approach.

The Place of In Vitro Maturation in PCO/PCOS.

In vitro maturation (IVM) of human oocytes is an emerging treatment option for women with polycystic ovary/polycystic ovary syndrome (PCO/PCOS) in addition to the standard in vitro fertilization (IVF) treatment. There has been significant improvements in pregnancy rates with IVM over the last two decades. This article reviews the place of IVM for women with PCO/PCOS, placing an emphasis on the predictors of successful pregnancy, optimization of culture media, IVM protocols, pregnancy rates, and neonatal outcomes following IVM treatment.

Oocyte in vitro maturation: A sytematic review.

In vitro maturation (IVM) is one of the most controversial aspects of assisted reproductive technology. Although it has been studied extensively, it is still not a conventional treatment option and is accepted as an alternative treatment. However, studies have shown that IVM can be used in almost all areas where in vitro fertilization (IVF) is used and it has a strong place in fertility protection and Ovarian Hyperstimulation syndrome management. The aim of this systematic review was to address all aspects of the current knowledge of IVM treatment together with the evolution of IVM and IVF.

Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes.

High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets’ content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses.

Human chorionic gonadotrophin priming for fertility treatment with in vitro maturation.

In vitro maturation (IVM) is a fertility treatment that involves the transvaginal retrieval of immature oocytes, and their subsequent maturation and fertilisation. Although the live birth rate is lower than conventional in vitro fertilisation (IVF) with ovarian stimulation, it is a useful treatment, as it avoids the risk of ovarian hyperstimulation syndrome (OHSS). Women with polycystic ovaries (PCO) or polycystic ovarian syndrome (PCOS) are at an increased risk of OHSS. Thus, IVM may be a more useful treatment in this patient group.Strategies to maximise the maturation rates of the immature oocytes are important. This review focuses on the administration of human chorionic gonadotrophin (hCG) prior to immature oocyte retrieval. To determine the effectiveness and safety of hCG priming in subfertile women who are undergoing IVM treatment in the context of assisted reproduction. We searched the following electronic databases up to 29 August 2016: Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials, CENTRAL, MEDLINE, Embase, PsycINFO, and CINAHL. We also searched the trial registries ClinicalTrials.gov and WHO ICTPR to identify ongoing and registered trials. We sought recently published papers not yet indexed in the major databases, and reviewed the reference lists of reviews and retrieved studies as sources of potentially relevant studies. There were no language restrictions. We included randomised controlled trials (RCTs) that compared hCG priming with placebo or no priming in women undergoing IVM. We also included RCTs that compared different doses of hCG, or the timing of oocyte retrieval. The primary outcomes were live birth rate and miscarriage rate per woman randomised. Two review authors independently selected studies for inclusion, and with a third author, assessed risk of bias and extracted data. We contacted the original authors where data were missing. For dichotomous outcomes, we used the Mantel-Haenszel method to calculate odds ratios (OR). For continuous outcomes, we calculated the mean differences (MD) between treatment groups. We assessed statistical heterogeneity using the I² statistic. We assessed the overall quality of the evidence using GRADE methods. We included four studies, with a total of 522 women, in the review. One of these studies did not report outcomes per woman randomised, and so was not included in formal analysis. Three studies investigated 10,000 units hCG priming compared to no priming. One study investigated 20,000 units hCG compared to 10,000 units hCG priming. Three studies only included women with PCOS (N = 122), while this was an exclusion criteria in the fourth study (N = 400).We rated all four studies as having an unclear risk of bias in more than one of the seven domains assessed. The quality of the evidence was low, the main limitations being lack of blinding and imprecision.When 10,000 units hCG priming was compared to no priming, we found no evidence of a difference in the live birth rates per woman randomised (OR 0.65, 95% confidence intervals (CI) 0.24 to 1.74; one RCT; N = 82; low quality evidence); miscarriage rate (OR 0.60, 95% CI 0.21 to 1.72; two RCTs; N = 282; I² statistic = 21%; low quality evidence), or clinical pregnancy rate (OR 0.52, 95% CI 0.26 to 1.03; two RCTs, N = 282, I² statistic = 0%, low quality evidence). Though inconclusive, our findings suggested that hCG may be associated with a reduction in clinical pregnancy rates; 22% of women who received no priming achieved pregnancy, while between 7% and 23% of women who received hCG priming did so.The study comparing 20,000 units hCG with 10,000 units hCG did not report sufficient data to enable us to calculate odds ratios.No studies reported on adverse events (other than miscarriage) or drug reactions. This review found no conclusive evidence that hCG priming had an effect on live birth, pregnancy, or miscarriage rates in IVM. There was low quality evidence that suggested that hCG priming may reduce clinical pregnancy rates, however, these findings were limited by the small number of data included. As no data were available on adverse events (other than miscarriage) or on drug reactions, we could not adequately assess the safety of hCG priming. We need further evidence from well-designed RCTs before we can come to definitive conclusions about the role of hCG priming, and the optimal dose and timing.

Immature oocyte retrieval and in vitro oocyte maturation at different phases of the menstrual cycle in women with cancer who require urgent gonadotoxic treatment

To evaluate the feasibility and the efficacy of invitro maturation (IVM) when immature oocyte collection was performed in the early follicular, late follicular, or luteal phases in women with cancer who require urgent chemotherapy. Retrospective cohort study. University teaching hospital. One-hundred and sixty-four women with cancer undergoing IVM treatment for fertility preservation. Oocyte retrieval, IVM, cryopreservation. Medians (interquartile range) of oocytes collected, maturation rates after 48hours of culture, and metaphase II oocytes cryopreserved. The analysis included a total of 192 cycles grouped into early follicular phase (n = 46), late follicular phase (n = 107), or luteal phase (n = 39). Embryo cryopreservation was performed in 82 cycles, and oocyte cryopreservation in 105 cycles. Between the early follicular, late follicular, and luteal phases, no statistically significant differences were found in the number of oocytes collected (8.5 [4-15.8], 8 [5-14], and 7 [4-9], respectively), the maturation rates after 48hours of culture (53.5% [39.8-77], 58% [44-82], and 50% [33-67], respectively), or the number of oocytes cryopreserved (3 [0-7.3], 3 [0-7], and 3 [1-5.5], respectively). Similarly, the fertilization rates (77 [62.8-92.5], 75 [60-100], and 63.5 [50-75], respectively) and number of embryos cryopreserved (3 [2-5.8], 3 [0.5-5], and 2 [1-3], respectively) were not statistically significantly different among the groups. Our study confirms the feasibility of IVM collection at any time during the menstrual cycle. Treatment with IVM is an alternative method when chemotherapy cannot be delayed or ovarian stimulation is contraindicated. The long-term outcomes remain to be studied.

Does in vitro maturation (IVM) have an impact on morphokinetic parameters in comparison with IVF?

Polycystic ovarian syndrome (PCOS) patients are at higher risk of ovarian hyper stimulation syndrome (OHSS) due to the high dose of gonadotropin in IVF. To avoid the possibility of OHSS, IVM techniques have been developed and employed in clinical settings. However, IVM delivers lower pregnancy rates and birth rates than IVF. The objective of this study was to assess whether IVM treatment affects embryo development events together with morphokinetic parameters compared with IVF, and to identify the morphokinetic parameters specific to embryos that were suitable for implantation in IVM cycles. Retrospective study. The subjects were 41 couples who underwent 50 cycles of IVM at our clinic, from May, 2013 to Nov., 2015. After the failure of IVM treatment, 12 couples underwent standard IVF treatment. We assessed 194 embryos derived from IVM-ICSI and 48 embryos derived from IVF-ICSI. Their treatment outcomes were analyzed with morphokinetic events (cell division and interval of cell cleavage) by Primovision (vitrolife Sweden), and rate of clinical outcome. Moreover in IVM cycles, these time points were also compared between transferred embryos that implanted or did not implant. IVM zygotes showed significantly lower good quality embryo on day3, blastocyst formation, and good blastocyst rates compared with IVF. There were no differences in time points of cell division (tPNa to t8), pregnancy and abortion rates between the two groups. However, there were significant differences in synchrony of the second cell cycles (t4-t3:S2) (S2:4.5±5.0h vs. 1.4±2.3h, P=0.0002), and the rate of direct cleavage of one to three, or there to five cells was significantly higher in the IVM group (90/194 46.4% vs. 9/48 18.8%, P=0.0016). In the IVM group, there were no differences in development time points between implanted and non-implanted embryos. IVM zygotes showed increased abnormal cleavage compared with IVF; however, there were no significant differences in pregnancy rate or abortion rate between IVM and IVF. We rejected these abnormal cleaved embryos for transfer, indicating that in order to improve the pregnancy rate it is important to exclude the abnormal cleaved embryo, such as direct cleavage, from embryo transfer in IVM treatment.

Outcomes of in vitro fertilization cycles among patients with polycystic ovary syndrome following ovarian puncture for in vitro maturation

ObjectiveTo investigate the effects of ovarian puncture for in vitro maturation (IVM) on subsequent in vitro fertilization (IVF) embryo transfer cycles in patients with polycystic ovary syndrome (PCOS). MethodsA retrospective study included data from patients admitted to the First Affiliated Hospital of Wenzhou Medical University, China, between January 1, 2008 and December 31, 2014. Patients with PCOS undergoing IVF cycles after having been treated with IVM unsuccessfully were included as the study group and an IVF-procedure data-matched control group of patients undergoing their first IVF cycles was included in a 1:4 ratio. Patients with reproductive anomalies were excluded. Endocrine-hormone levels and antral follicle counts were measured and fertilization-related outcomes were evaluated. ResultsThere were 49 patients included in the study group and 196 included in the control group. Within the study group, basal luteal-hormone, testosterone, and antral follicle count levels were significantly lower following IVM treatment. The total gonadotropin dose was lower (P<0.001) and the duration of stimulation was shorter (P<0.001) in the study group compared with the control group. The clinical-pregnancy rate was higher in the study group (P=0.018) and no difference was observed between the groups in ovarian hyper-stimulation syndrome (P=0.633). ConclusionsPrevious IVM resulted in improved endocrine profiles and increased clinical-pregnancy rates among patients with PCOS undergoing IVF cycles.

Application of serum anti-Müllerian hormone levels in selecting patients with polycystic ovary syndrome for in vitro maturation treatment

ObjectiveThe purpose of this study was to identify useful clinical factors for the identification of patients with polycystic ovary syndrome (PCOS) who would benefit from in vitro maturation (IVM) treatment without exhibiting compromised pregnancy outcomes.MethodsA retrospective cohort study was performed of 186 consecutive patients with PCOS who underwent human chorionic gonadotropin-primed IVM treatment between March 2010 and March 2014. Only the first IVM cycle of each patient was included in this study. A retrospective case-control study was subsequently conducted to compare pregnancy outcomes between IVM and conventional in vitro fertilization (IVF) cycles.ResultsThrough logistic regression analyses, we arrived at the novel finding that serum anti-Müllerian hormone (AMH) levels and the number of fertilized oocytes in IVM were independent predictive factors for live birth with unstandardized coefficients of 0.078 (95% confidence interval [CI], 1.005–1.164; p=0.037) and 0.113 (95% CI, 1.038–1.208; p=0.003), respectively. Furthermore, these two parameters were able to discriminate patients who experienced live births from non-pregnant IVM patients using cut-off levels of 8.5 ng/mL and five fertilized oocytes, respectively. A subsequent retrospective case-control study of patients with PCOS who had serum AMH levels ≥8.5 ng/mL showed that IVM had pregnancy outcomes comparable to conventional IVF, and that no cases of ovarian hyperstimulation syndrome were observed.ConclusionSerum AMH levels are a useful factor for predicting pregnancy outcomes in PCOS patients before the beginning of an IVM cycle. IVM may be an alternative to conventional IVF for PCOS patients if the patients are properly selected according to predictive factors such as serum AMH levels.

In vitro maturation is associated with increased early embryo arrest without impairing morphokinetic development of useable embryos progressing to blastocysts.

Does polycystic ovarian syndrome (PCOS) or in vitro maturation (IVM) treatment affect embryo development events and morphokinetic parameters after time-lapse incubation? There was an increase in some abnormal phenotypic events in PCOS-IVM embryos as well as an increase in early arrest of PCOS-IVM and PCOS-ICSI embryos; however, IVM treatment or PCOS status did not alter morphokinetic development of embryos suitable for transfer of vitrification. IVM has been less successful than standard IVF in terms of clinical pregnancy, implantation and live birth rates. There is currently no information available about the development of IVM embryos according to time-lapse analysis. This article represents a prospective case-control study. The study involved 93 participants who underwent 93 treatment cycles. Cycles were completed between January 2013 and July 2014. Participants were recruited for the study at Fertility Specialists of WA and Fertility Specialists South, Perth, Western Australia. Of the PCOS diagnosed patients, 32 underwent IVM treatment (PCOS-IVM) and 23 had standard ICSI treatment (PCOS-ICSI). There were 38 patients without PCOS who underwent standard ICSI treatment comprising the control group (control-ICSI). The PCOS-IVM group showed significantly more embryos with multinucleated two cells (P = 0.041), multinucleated four cells (P = 0.001) and uneven two cells (P = 0.033) compared with the control-ICSI group, but not the PCOS-ICSI group. There were no significant differences in the rates of any abnormal events between the PCOS-ICSI and control-ICSI groups. Embryo arrest between Days 2 and 3 was higher in the PCOS-IVM and PCOS-ICSI groups compared with the control-ICSI group (P < 0.001 and P = 0.001). Embryo arrest from Days 3 to 4 was higher in the PCOS-IVM group compared with both the PCOS-ICSI and control-ICSI groups (P < 0.001). There were no differences in embryo arrest rates across all three groups at the compaction or blastulation stages. Cumulative rates of embryo arrest, from the time to second polar body extrusion (tPB2) to the time to formation of a blastocyst (tB), result in a decreased proportion of useable PCOS-IVM blastocysts compared with the other two treatment groups; however, of the embryos remaining, there was no significant difference in morphokinetic development between the three groups. This was a small study using time-lapse analysis of embryo development as the primary end-point. Larger, randomized, clinical trials are required to clarify the implications of time-lapse incubation of IVM embryos and the effects on implantation and ongoing pregnancy. This is the first study to compare the time-lapse analysis of IVM with standard ICSI for patients with and without PCOS. This allows for a more detailed and specific timeline of events from embryos generated using this approach for patients diagnosed with PCOS and shows that embryos generated from IVM have an increased rate of early embryo arrest, however; morphokinetic development is not impaired in embryos that progress to the useable blastocyst stage. The study was supported by the Women's and Infant's Research Foundation of Western Australia. R.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. The other authors have no competing interests.

Human cumulus-enclosed germinal vesicle oocytes from early antral follicles reveal heterogeneous cellular and molecular features associated with in vitro maturation capacity.

Are oocyte size, chromatin remodelling, transcriptional activity and mitochondrial distribution in human immature oocytes from early antral follicles retrieved for in vitro maturation (IVM) associated with the acquisition of meiotic competence? Oocyte size, chromatin compaction, cessation of RNA synthesis and mitochondria rearrangement around the nucleus are associated with the oocyte's potential to resume meiosis in vitro. Information on oocyte features that confer meiotic competence in human mainly derives from germinal vesicle (GV) oocytes that failed to resume meiosis following an hCG trigger after ovulation induction cycles. Characterization of cumulus-enclosed GV oocytes from small antral follicles prior to IVM provides knowledge on the initial oocyte status and suggests culture requirements in order to promote oocyte competence in vitro. Prospective collection of 107 oocytes immediately after retrieval (before IVM) and of 293 GV oocytes that had failed to resume meiosis (after IVM). Human oocytes were collected from women with polycystic ovary syndrome (PCOS), receiving in total 450 IU of highly purified-hMG for IVM treatment (patients) or who donated oocytes for IVM research (donors). Oocytes at GV-stage were retrieved from follicles <10 mm (range 2-10 mm) diameter, before IVM (oocytes at retrieval) or those that failed to mature after IVM (meiotically incompetent). Oocytes were allocated for either mitochondrial staining, by incubating in mitotracker red and then fixed; or for nascent RNA staining, which was assessed by fluorescent labelling (Click-iT(®) RNA Assay). In every case, oocyte diameter was recorded and chromatin was stained after oocyte fixation. GV-stage oocytes were analysed by confocal laser-scanning microscopy and their characteristics were compared and related to their meiotic competence. Analysis of oocytes at the immature GV-stage revealed that oocytes at retrieval were significantly larger than those that failed to resume meiosis after IVM (112.7 versus 109.6 µm, P < 0.0001). Oocytes assessed at retrieval showed that 50.6% had a condensed chromatin configuration (perinucleolar chromatin rim) and were consistently transcriptionally silent. This rate matched maturation rates in our current in vitro culture system (49%). However, oocytes that had not reinitiated meiosis after 30 h IVM demonstrated, apart of being smaller in diameter, significantly higher rates of dispersed or intermediate chromatin (P = 0.005). Analysis of mitochondrial distribution revealed that many oocytes at retrieval displayed mitochondrial internalization towards the nucleus (12/30) or a perinuclear mitochondrial distribution (6/30). These mitochondrial patterns were observed more rarely in GV incompetent oocytes following 30 h IVM (16/98 and 1/98, respectively). Most of the analyses involved the use of invasive techniques. Hence, despite the fact that these data deliver essential information on the intrinsic oocyte maturational and developmental status, a direct match with embryological outcomes could not be established. The evidence described here can aid in tailoring IVM systems in order to promote completion of nuclear and cytoplasmic maturation of unexpanded cumulus-oocyte complexes. This study was supported by research grants by the Institute for the Promotion of Innovation by Science and Technology in Flanders, project numbers IWT 130327 and 110680; the Fund for Research Flanders, project number FWO G.0343.13, the Belgian Foundation Against Cancer (HOPE project) and COOK Medical. None of the authors has any competing interest to declare.

Embryos from in Vitro Maturation (IVM) Technique Can Be Successfully Vitrified Resulting in the Birth of a Healthy Child

IVM can be an advantageous technique when applied to PCOS (Polycystic Ovarian Syndrome) patients. The oocytes are retrieved from antral follicles of non-stimulated ovaries, specially preventing hyperstimulation syndrome. Apart from its role as a reproductive treatment, IVM has emerged as a promising tool for emergency fertility preservation, since it can be performed flexibly in either follicular or luteal phase. A 34-year-old patient with PCOS, high body mass index and tubal factor was submitted twice to IVM treatment. Her husband has low count spermatozoa. The first IVM cycle was in 2009, she transferred 3 fresh embryos and got pregnant giving birth to a healthy boy weighing 3.3 kg. In 2013, the patient returned for another IVM cycle and the embryos had to be vitrified because she failed to develop an adequate endometrium for transfer. In the next cycle, the endometrium was prepared using estrogen and progesterone and the two best embryos were warmed up and transferred. She became pregnant and after 36 weeks gave birth to a healthy girl weighing 2.7 kg. She still has four embryos left to transfer. IVM may be an alternative technique to be considered when dealing with PCOS patients. Although clinical outcomes are currently inferior when compared with conventional hormone driven ART (Artificial Reproductive Techniques), it does apply in some cases while preventing hyperstimulation risks. Thus, embryos obtained by IVM can also be vitrified with successful outcomes.

134 SERIAL TREATMENT OF RESVERATROL-TROLOX IMPROVED EMBRYONIC DEVELOPMENT OF PORCINE PARTHENOTES

Antioxidants are widely used for in vitro production of embryos due to their activity as reactive oxygen species scavengers. Among various antioxidants, resveratrol supplementation in in vitro-maturation (IVM) media and trolox supplementation in in vitro-culture (IVC) media improves oocyte maturation and embryonic development in other species, such as cattle and sheep. Limited information is available, however, on the effect of resveratrol and/or trolox on porcine embryos produced in vitro. In this study, we evaluated the effect of resveratrol supplemented to the media of IVM and trolox treatment during IVC on porcine parthenotes. We used TCM-199 as IVM media and porcine zygote medium (PZM)-5 as IVC media. For activation, matured oocytes after 44 h of IVM were electrically activated with 280 mM mannitol and cultured in IVC medium (PZM-5). Statistical analyses of all data were carried out using SPSS 17.0 (one-way ANOVA, followed by Duncan's multiple range test). In the experiment 1, a total of 618 oocytes were used in 4 independent replicates to evaluate the effect of 4 different concentrations (0, 1, 2, or 4 μM) of resveratrol during IVM on parthenotes. Oocytes treated with 2 μM resveratrol during IVM had significantly higher cleavage rates and blastocyst formation rates (73.0 and 34.4% v. 64.0 and 18.3%, respectively) than the control group. Experiment 2 involved supplementation with trolox (0 μM, 100 μM, 200 μM, 400 μM) to 957 parthenotes during IVC for 7 days (4 replicates). Cleavage rates significantly increased in the 100 μM group (75.6 v. 69.1%), and blastocyst formation rates in the 200 μM group were significantly higher compared to the control group (33.7 v. 23.8%). To determine the combined effects of resveratrol treatment during IVM and trolox treatment during IVC, in the experiment 3 we selected an optimized concentration (2 μM of resveratrol and 200 μM of trolox) from each experiment and evaluated the combined effects (3 times replicated). We designed 4 groups: (1) control, (2) resveratrol only (R), (3) trolox only (T), and (4) resveratrol-trolox (R-T). The R group and R-T group showed significantly higher cleavage rates than the control group (81.8 and 83.1% v. 72.3%). All treatment groups showed significantly increased blastocyst formation rates compared with the control group (39.2, 37.8, and 38.4% v. 23.7%). There is no significant difference in total cell numbers of blastocyst among the control, R, and T groups (47.8 v. 54.2 v. 54.7). However, the R-T group had significantly more cells than the control group (67.1 v. 47.8). Our results suggest that 2 μM resveratrol treatment during IVM, followed by 200 μM trolox treatment during IVC, improves developmental potential of the parthenotes. For a further study, we will apply this condition to somatic cell nuclear transfer, and we also will verify quantitative PCR analysis of apoptosis-related mRNA expression of PA and somatic cell nuclear transfer embryos. This study was supported by the MOTIE (#10033839), IPET (#311011-05-3-SB010), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

285 REGULATION OF OOCYTE MEIOTIC RESUMPTION USING cAMP MODULATORS IN BOVINE IN VITRO MATURATION

In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP). Currently, IVP has low efficiency due to an inadequate IVM system where premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3′,5′-cyclic adenosine monophosphate (cAMP). Some successful methods of improving IVM have utilised cAMP modulators to maintain high intraoocyte cAMP, delaying the onset of nuclear maturation and allowing cytoplasmic maturation to occur. The current experiment is a follow-up to previous work in which an extended 2-step maturation system was examined. In the previous experiments, we found that meiotic resumption was significantly delayed, but the overall maturation rates of extended IVM were about half those of standard IVM, suggesting that the effects of the modulators on the oocytes were not completely reversible. The current experiment compares cAMP concentrations of oocytes in this extended IVM to standard IVM to determine whether high cAMP is the cause of the low maturation rate. Bovine oocytes (n = 686) were obtained from mixed-breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into 2 groups: standard IVM and extended IVM. Standard IVM consists of a 23-h maturation composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured at 39°C in 5% CO2. Extended IVM consists of 2 steps: a pre-IVM phase composed of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 h at 39°C, followed by an extended IVM phase composed of standard IVM media supplemented with 20 µM cilostamide for 31 h (39°C, 5% CO2). Additionally, oocytes in the extended IVM treatment group where held in HEPES-TALP media with FSK and IBMX during the 2-h oocyte collection period in order to prevent any decrease in cAMP before the oocytes could be placed in the extended IVM media. Oocytes in standard IVM were sampled at 0, 8, and 23 h of maturation, while oocytes in extended IVM were sampled at 0, 8, 18, and 33 h of maturation. Cumulus cells were removed from all oocytes by vortexing in hyaluronidase solution. Oocytes were stored in groups of 10 at –80°C. A cAMP enzyme immunoassay (GE Healthcare) was performed to determine cAMP concentrations throughout standard and extended IVM. Assay results were analysed using an ANOVA followed by a Tukey's pairwise test (Sigma Stat 3.5) to detect significant differences (P &lt; 0.05). Results indicated significantly higher cAMP levels in extended IVM oocytes during the first 3 h after collection using FSK and IBMX in the holding media (0.505 v. 1.006 fmol/oocyte, P = 0.035) but cAMP levels were not maintained in the cilostamide-only extended IVM medium. This suggests that high cAMP levels were not the cause of low maturation rates in extended IVM, since cAMP concentrations did decrease after 3 h. Possible negative effect of cilostamide on these oocytes may be suggested and need to be analysed.