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Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.

Background The addition of a GnRH analogue to the luteal phase in in vitro fertilization programs has been seldom proposed due to the presence of GnRH receptors in the endometrium. The aim of the study was to evaluate the effect of triptorelin addition in short antagonist cycles, compared to cycles where the only supplementation was progesterone. Methods The primary objective of this study was the study of the effect of Triptorelin addiction during the luteal phase on the live birth rate. Secondary objectives of efficacy were pregnancy rates and implantation rates, as well as safety in terms of OHSS risks. The study was a prospective, randomized, open study, performed in two independent Centers from July 2013 to October 2015. Patients were divided into three groups: a) Regular antagonist protocol, with only luteal progesterone; b) Antagonist protocol with luteal triptorelin as multiple injections, c) Antagonist protocol with luteal triptorelin as single bolus. Descriptive statistics were obtained for all the parameters. Mean and standard deviation were used for all quantitative parameters. Differences between percentages were studied using Chi-square test generalized to the comparison of several proportions. Results A total number of 1344 patients completed the study, 786 under the age of 35 years, and 558 over 35 years. It was observed an increase of positive HCG results, Clinical pregnancy rates and Delivery rates when triptorelin was added in the luteal phase, irrespective whether as a single bolus or five injections. This increase was statistically significant both for pregnancy rates and delivery rates. The statistic difference between pregnancies and deliveries obtained with or without luteal triptorelin reached p  < 0,01. No increase of OHSS risk was observed. Conclusions From this large study it appears that the concept of luteal phase supplementation should be revisited. From our study it appears that triptorelin addition to the luteal phase of antagonist cycles, either as a single bolus or using multiple injections, is a good tool to optimize ART results. Trial registration The study was approved by the Ethics Committee of Provincia di Bergamo (n 1203/2013).

Given the importance of embryo developmental competence assessment in reproductive medicine and biology, the aim of this study was to compare the performance of fertilization and cleavage morphokinetics with embryo morphology to predict post-ICSI live birth. Data from embryos cultured in a time-lapse microscopy (TLM) incubator and with known live birth outcomes (LB: embryos achieving live birth, n = 168; NLB: embryos not achieving live birth, n = 1633) were used to generate receiver operating characteristic (ROC) curves based on morphokinetic or morphological scores, and the respective areas under the curve (AUC) were compared. The association between live birth and 12 combinations of four morphokinetic quality degrees (A–D) with three morphological quality degrees (A–C) was assessed using multivariate analysis. Morphokinetic parameters from tPNa to t8 were reached earlier in LB compared with NLB embryos. The ROC curve analysis indicated that morphokinetic information is more accurate than conventional morphology to predict live birth [AUC = 0.64 (95% CI 0.58–0.70) versus AUC = 0.58 (95% CI 0.51–0.65)]. The multivariate analysis was in line with AUCs, revealing that embryos with poor morphokinetics, independently of their morphology, provide lower live birth rates ( P < 0.001). A considerable percentage of embryos with top morphology presented poor morphokinetics (20.10%), accompanied by a severely reduced live birth rate in comparison with embryos with top morphology and morphokinetics ( P < 0.001). In conclusion, TLM-derived early morphokinetic parameters were more predictive of live-birth achievement following ICSI than conventional morphology.

Purposes The aim of this study is to determine whether a clinical advantage is gained with use of LH in combination with FSH or as a component of human menopausal gonadotropin (hMG) to achieve optimal ovarian stimulation. Methods In this study, we compared retrospectively two regimens, r-FSH/r-LH and hMG, for the treatment of women with reduced ovarian reserve, identified as subjects with antral follicle count (AFC) < 11 and AMH ≤ 1.1 ng/ml. Results Overall, the clinical pregnancy per started cycle was higher in the r-FSH/r-LH group (12.5 vs. 8.1%, P  < 0.02), while implantation (11.1 vs. 9.5%) and miscarriage rates (29.9 vs. 35.9%) were comparable. Data were further analysed performing separate comparisons in subpopulations with different ranges of AFC, i.e. < 4, 4–6 and 7–10. Major differences between the two regimens were observed in women with AFC < 4. In this subpopulation, not only was the clinical pregnancies per started cycle higher in the r-FSH/r-LH group (10.2 vs. 1.5%, P  < 0.01), but also implantation was significantly higher (13.0 vs. 2.8%, P  < 0.02). Conclusions A r-FSH/r-LH regimen appears to be beneficial for the treatment of women with extremely poor ovarian reserve. It should be considered however that, being retrospective, this study is affected by obvious limitations, such as post-treatment patient selection criteria and absence of randomisation.

Purpose In in vitro maturation (IVM) cycles primed with human chorionic gonadotropin (hCG), both immature and mature oocytes are retrieved from antral follicles sized 8–12 mm. Using time-lapse microscopy, we compared the morphokinetic behavior of embryos developed from oocytes matured in vivo and in vitro, testing the hypothesis that IVM affects preimplantation development. Furthermore, we extended the morphokinetic analysis of these embryos by a comparison with embryos obtained in stimulated assisted reproduction technology (ART) cycles. Methods In IVM cycles primed with follicle-stimulating hormone (FSH)/hCG, prior to sperm microinjection, oocytes surrounded by an expanded cumulus at retrieval and presumably mature (EC-MII) were incubated for 6 h, while immature oocytes enclosed in a compact cumulus (CC) were matured in vitro for 30 h. The morphokinetics of embryos selected for transfer or cryopreservation, derived from EC-MII and CC oocytes, were comparatively and retrospectively analyzed in terms of cleavage times (t2, t3, t4, t5, and t8) and intervals (cc2, cc3, s2, s3). For further comparison, the morphokinetics of embryos selected for transfer or cryopreservation (ICSI) or giving rise to ongoing pregnancies (model) in stimulated ART cycles was also assessed. Results The morphokinetic behavior of EC-MII and CC embryos was entirely comparable, as suggested by the absence of statistical differences in the averages of all cleavage times and intervals. Almost all cleavage and interval times were also similar between EC-MII, CC, ICSI, and model groups, with the exception of t4 and s2, which were delayed and longer, respectively, in embryos generated in IVM cycles (EC-MII and CC). Conclusions These findings do not support the hypothesis that maturation in vitro affects embryo morphokinetics, while they suggest only marginal differences in the morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles and embryos developed from mature oocytes recovered in stimulated cycles.

Purpose To assess retrospectively the developmental potential of different types of cumulus cell-oocyte complexes (COCs) derived from IVM cycles. Methods IVM cycles were performed in natural cycles or after HCG, FSH, or FSH/HCG priming. COCs recovered were morphologically characterized in different types: compact (CC) or expanded (EC) cumulus mass but including an immature oocyte, and expanded cumulus mass enclosing a mature oocyte (EC-MII). Embryo developmental competence was investigated analysing exclusively cycles in which all transferred embryos derived from the same COC category. Results Fertilization rates did not differ significantly. Significant differences in pregnancy rates (14.5 %, 10.0 % and 27.6 % in the CC, EC, and EC-MII categories, respectively) were observed. Likewise, significant differences in implantation rates (8.9 %, 6.3 % and 19.1 % in the CC, EC, and EC-MII categories, respectively) were found. Overall, priming with FSH/HCG had a beneficial effect on pregnancy and implantation rates, while no priming or HCG alone generated oocytes with poor competence. Conclusions In IVM cycles, morphological evaluation at the time of collection can predict the developmental ability of different COCs. FSH/HGC priming has a positive effect on oocyte competence.

Purpose The aim of this retrospective study was to compare the competence of oocytes obtained from preovulatory and antral follicles. Methods Mature oocytes from preovulatory follicles were retrieved from women selected for standard IVF treatment (Group A). Mature oocytes from antral follicles were recovered from women undergoing hCG-primed in vitro maturation (IVM) treatment (Group B). Patients groups were matched for age, BMI, FSH, AMH and antral follicle count (AFC) values. In vivo matured oocytes from both groups were microinjected and resulting embryos were culture and selected on day 3 for embryo transfer. Results Oocyte pick-ups (OPU) were 315 and 204 in Groups A and B, respectively. Fertilization rates were comparable (72.8 and 75.9 %, respectively; P  = 0.137). In Group A, in which the average number of embryos transferred was higher, clinical pregnancy rates per OPU (37.5 %) and embryo transfer (38.4 %) were superior in comparison to Group B (27.0 %, P  = 0.013; 29.4 %, P  = 0.041; respectively). On the other hand, implantation rates (Group A, 23.7 %; Group B, 20.8 %) and proportions of babies born per transferred embryo (Group A, 19.5 %; Group B, 16.9 %) were similar ( P  = 0.528 and 0.332, respectively). Conclusions Overall, this suggests that oocyte competence is already achieved at the antral stage of follicle development.

Purpose In oocyte in-vitro maturation (IVM) treatments, the chances to achieve a pregnancy are critically dependent on the retrieval of a suitable number of oocytes. In this study, we assessed the ability of circulating levels of anti-mullerian hormone (AMH) to identify normo-ovulatory women suitable for IVM treatment on the basis of the number of retrieved oocytes. Method Serum AMH was quantified in normo-ovulatory women younger than 39 years undergoing IVM treatment. After immature oocyte retrieval and IVM, maximum 3 mature oocytes were used for treatment and all resulting embryos were transferred, as established by law. From 177 cycles, 991 oocytes were recovered. Following IVM, 484 mature oocytes were obtained (50.1%). Results The overall pregnancy rate per embryo transfer was 16.6% (25/151) and the implantation rate was 10.9% (30/278). Linear regression and receiver operating characteristic (ROC) analyses were applied to identify independent variables and quantify a cut-off AMH value able to identify patients suitable for IVM treatment. An AMH value of 1.28 ng/ml was identified as a threshold for the prediction of the retrieval of at least 5 oocytes, with a sensitivity of 93.4% and a specificity of 33.8%. Positive and negative predictive values were 67.6% and 75.0%, respectively. Conclusions AMH can be adopted to identify women candidate for an IVM treatment from whom a suitable number of oocytes may be retrieved. This is of crucial significance during a non-stimulated cycle, in order to prevent an insufficient oocyte collection and rescue the treatment by implementing a conventional controlled ovarian stimulation.

Purpose Only 50–60 % of immature human oocytes attain the mature stage in vitro. Such a deficiency may be a reflection of inadequate conditions of in vitro maturation (IVM) or a manifestation of intrinsic oocyte defects. In the present study, we explored the possibility that the DNA of immature oocytes may be damaged and that such a condition, or inability to trigger a repair action, is associated to germinal vesicle (GV) arrest. Methods Immature oocytes (GV-stage oocytes) were obtained from women undergoing stimulated (Stim-C) or IVM (IVM-C) cycles. GV oocytes obtained from stimulated cycles were fixed for successive analysis either after recovery (T0) or following 30 h (T30) of culture if still arrested at the GV stage. Oocytes retrieved in IVM cycles were used only if they were found arrested at the GV stage after 30 h (T30) of culture. All oocytes were fixed and stained to detect chromatin and actin. They were also assessed for positivity to γH2AX and Rad51, markers revealing the presence of double-strand DNA breaks and the activation of a DNA repair response, respectively. Labelled oocytes were analysed using a Leica TCS SP2 laser scanning confocal microscope. Results In Stim-C oocytes, γH2AX positivity was 47.5 and 81.5 % in the T0 and T30 groups, respectively ( P  = 0.003), while γH2AX-positive oocytes were 58.3 % in the IVM-C T30 group (Stim-C T0 vs. IVM-C T30, P  = 0.178; Stim-C T30 vs. IVM-C T30, P  = 0.035). Positivity for nuclear staining to Rad51 occurred in 42.1 and 74.1 % of Stim-C in the T0 and T30 subgroups, respectively ( T  = 0.006), while 66.7 % of IVM-C T30 oocytes resulted positive for a DNA repair response (Stim-C T0 vs. IVM-C T30, P  = 0.010; Stim-C T30 vs. IVM-C T30, P  = 0.345). Conclusions The present data document the existence of double-strand DNA breaks (DSBs) in human immature oocytes. Also, they are consistent with the hypothesis that insults to DNA integrity may be an important factor affecting meiotic resumption.