GnRH agonists to sustain the luteal phase in antagonist IVF cycles: a randomized prospective trial.

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).

The first report of pregnancies following blastocyst automated vitrification in Europe.

Embryo cryopreservation is a valuable technique in assisted reproductive technology (ART) that increases cumulative pregnancy rates and allows postponement of embryo transfer in patients with undesirable uterine or clinical conditions. Although vitrification has been considered the most efficient method to freeze oocytes and embryos, it is time-consuming and highly operator-dependent. Gavi® is the first semi-automated machine for vitrification capable of controlling crucial variables such as temperature, volume, concentration and exposure time during the vitrification process. We report the first two pregnancies obtained with blastocysts cryopreserved with the Gavi® semi-automated vitrification system in Europe. These outcomes suggest that the utilization of semi-automated vitrification may contribute to improve the outcomes and laboratory logistics of fertility clinics.

From a circle to a sphere: the ultrasound imaging of ovarian follicle with 2D and 3D technology.

Ultrasound follicular count (antral follicle count, AFC) is a necessary tool for measuring ovarian reserve, whereby the estimated number of follicles responsive to FSH can predict the number of oocytes retrieved in IVF cycles and may be the basis for individualized ovarian stimulation therapy. Advances in the ultrasound technology have recently lead to the improvement in resolution and quality of the image. Moreover the automatic measurements of follicular diameter by using some specific 3D software seems associated to several advantages when compared to the 2D technique. Examination time is reduced because the ultrasound scan data are stored and can be analyzed in detail at a later time. These data can be reconstructed in any plane, regardless of the original scan plane facilitating the detailed analysis. Another advantage is that this new technique reduces the operator's influence on scan interpretation and objectivity; therefore, interobserver variability is reduced. Using follicular volume obtained with sono AVC as the measure of follicular growth combined with volume-based criteria for the hCG triggering may in the future improve the treatment outcome compared to that achieved with conventional monitoring with follicular diameter. Better knowledge in this area could be helpful to optimize IVF outcome, by refining ovarian stimulation protocols and obtain high quality oocytes.

Retrospective analysis of treatments with recombinant FSH and recombinant LH versus human menopausal gonadotropin in women with reduced ovarian reserve.

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.

Differential regulation of cumulus cell transcription during oocyte maturation in vivo and in vitro.

Differences in cumulus cell gene expression after oocyte maturation in vitro (IVM) or in vivo have been described in previous studies. However, the possible impact of follicle stage on gene expression deregulation during human oocyte IVM remains unknown. Expression of selected genes of interest was compared in cumulus cell of three classes of human cumulus cell-oocyte complexes (COCs): a) COCs derived from human chorionic gonadotropin (hCG)-triggered IVM cycles, collected at the germinal vesicle (GV) stage from mid-sized follicles (4-12 mm) and matured in vitro (IVM-GV); b) COCs derived from hCG-triggered IVM cycles, collected from mid-sized follicles (4-12 mm) and matured in vivo (IVM-MII); c) COCs derived from controlled ovarian stimulation in vitro fertilization (IVF) cycles, collected from large/preovulatory follicles and matured in vivo (IVF-MII). Overall, mRNA levels of the large majority of the 20 genes of different regulative and metabolic pathways subject to analysis were altered in IVM samples compared with in vivo matured COCs. In some cases, follicle size appeared to have a role in determining transcription deregulation. For example, in comparison to the IVF-MII control, the luteinizing hormone receptor was largely overexpressed in both IVM-GV and IVM-MII COCs, therefore irrespective of IVM. However, in other circumstances follicle size and IVM had distinct and opposite impacts on gene expression, as shown by transcription of amphiregulin, which was increased in IVM-MII COCs, but decreased in COCs matured in vitro (IVM-GV) compared with the IVF-MII control. This study confirms and extends previous data on gene expression dysregulation during IVM and indicates that the size of follicles from which immature oocytes are retrieved can be an independent factor of differential transcriptional regulation.

Dysmorphic patterns are associated with cytoskeletal alterations in human oocytes.

Study question: Are specific morphological anomalies in human mature oocytes, as revealed by transmitted light microscopy, associated with intrinsic damage to the meiotic spindle and actin cytoskeleton? Summary answer: Aggregates of smooth endoplasmic reticulum (SER) and domains of centrally localized granular cytoplasm (GC) reflect intrinsic damage to the oocyte cytoskeleton, namely alterations in spindle size, chromosome misalignment and cortical actin disorganization. What is known already: In preparation for ICSI, oocytes are often selected for use in treatment by morphological criteria, but the rationale and implications of this practice are controversial. Very little information is available on the relationship between oocyte morphology and intrinsic cellular characteristics, such as the actin cytoskeleton, meiotic spindle and chromosome alignment. Study design, size, duration: A total of 170 metaphase II (MII) oocytes were donated by consenting IVF patients and analysed; 62 were classified as morphologically normal (control), 54 had SER clusters and 54 had centrally localized GC. Participants/materials, setting, methods: Supernumerary oocytes were fixed within 3 h from recovery and stained for tubulin, chromatin and actin. Spindles were analysed for 1D and 2D characteristics by high-performance confocal microscopy. Chromosomes were classified as scattered or aligned and the conformation and intensity of cortical actin was evaluated. Main results and the role of chance: In comparison with control oocytes, both SER and GC oocytes showed greater spindle length (P = 0.033 and 0.003, respectively) and GC oocytes also showed greater spindle width (P= 0.049) and area (P= 0.036). Control and SER oocytes had statistically comparable rates of chromosome displacement from the metaphase plate, unlike GC oocytes where chromosome displacement occurred at higher rate (P = 0.013). In situations where a complete Z-stack was reconstructed from a polar angle, chromosome disposition was classified as being normal when two sets of concentric arrays were visible. Based on these parameters, the proportions of oocytes with normal chromosomal arrangement or partial/total disarrangement was not statistically different between control and SER oocytes. Conversely, in GC oocytes, chromosome disarrangement was higher (P = 0.002). All control oocytes displayed a continuous meshwork of suboolemmal actin, which appeared as an uninterrupted ring in thin optical sections. In contrast, in SER and  GC groups, integrity of suboolemmal actin was observed in only 66.7 and 42.9% of oocytes, respectively (P = 0.0001). Large scale data: N/A. Limitations reason for caution: Only two of several known oocyte dysmorphisms were investigated, while oocyte quality was assessed only by cytoskeletal criteria. Wider implications of the findings: This study represents a significant step toward a more objective assessment of oocyte morphology, offering information that can assist embryologists to make a more aware and rationally founded decision on whether, and with what possible implications, oocytes with certain dysmorphic characters should be used for treatment or discarded. More generally, it also demonstrates that morphometric parameters of the cytoskeleton and chromosome organization can be used as biomarkers of oocyte quality. Study funding and competing interest(s): This study was funded by Biogenesi Reproductive Medicine Centre (Monza, Italy). All authors declare no conflict of interests.

Ultrastructure of human oocytes after in vitro maturation.

STUDY HYPOTHESIS: How does the ultrastructure of human oocytes matured in vitro compare with oocytes collected from women after full hormonal stimulation? STUDY FINDING: The ultrastructure of human oocytes matured in vitro is largely, but not entirely, similar to those matured in vivo. WHAT IS KNOWN ALREADY: Embryos derived from in vitro-matured oocytes often have limited developmental potential, possibly as an effect of inappropriate in vitro maturation (IVM) conditions. Transmission electron microscopy (TEM) is a valuable research tool to compare in vivo and in vitro matured oocytes. However, previous studies on the ultrastructure of human IVM oocytes were done with inadequate material or inappropriate IVM conditions, and have limited significance. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Immature cumulus cell-enclosed oocytes, retrieved from mid-sized antral follicles of women requiring IVM treatment, were matured in vitro for 30 h. No leftover germinal vesicle-stage oocytes collected from fully stimulated cycles were used. Control in vivo matured oocytes were obtained from age-matched women undergoing full ovarian stimulation. In vitro and in vivo matured oocytes were analysed by TEM and compared according to previously established morphometric criteria of oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: All oocytes had normal ooplasm showing uniform distribution of organelles. Mitochondrial morphology appeared similar between the maturation conditions. Cortical granules were found typically stratified in a single, mostly continuous row just beneath the ooplasm in all oocytes. Microvilli were well preserved after IVM. Vacuoles were only occasionally found in all oocytes and, if present, they were frequently associated with lysosomes. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and mitochondria-vesicles (MV) complexes were commonly found in in vivo matured oocytes. However, large MV complexes partially replaced M-SER aggregates in IVM oocytes. LIMITATIONS, REASONS FOR CAUTION: As a note of caution it should be noticed that, being laborious and technically demanding, TEM cannot be applied to a large number of samples in a single investigation. Therefore, our data require further independent confirmation. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggests the notion that TEM remains a valuable research tool that can also offer quantitative data if associated with morphometric criteria of evaluation. Therefore, it can be adopted to test pre-clinically the performance of novel in vitro systems that are demanded to make oocytes IVM more successful in the human. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was independently funded by Biogenesi Reproductive Medicine Centre, Monza, Italy. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Morphokinetics of embryos developed from oocytes matured in vitro.

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.

Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption.

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.

Oocyte maturation: gamete-somatic cells interactions, meiotic resumption, cytoskeletal dynamics and cytoplasmic reorganization.

BACKGROUND: In a growth phase occurring during most of folliculogenesis, the oocyte produces and accumulates molecules and organelles that are fundamental for the development of the preimplantation embryo. At ovulation, growth is followed by a phase of maturation that, although confined within a short temporal window, encompasses modifications of the oocyte chromosome complement and rearrangements of cytoplasmic components that are crucial for the achievement of developmental competence. Cumulus cells (CCs) are central to the process of maturation, providing the oocyte with metabolic support and regulatory cues. METHODS: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning oocyte maturation in mammals. Searches were performed adopting 'oocyte' and 'maturation' as main terms, in association with other keywords expressing concepts relevant to the subject. The most relevant publications, i.e. those concerning major phenomena occurring during oocyte maturation in established experimental models and the human species, were assessed and discussed critically to offer a comprehensive description of the process of oocyte maturation. RESULTS: By applying the above described search criteria, 6165 publications were identified, of which 543 were review articles. The number of publications increased steadily from 1974 (n = 7) to 2013 (n = 293). In 2014, from January to the time of submission of this manuscript, 140 original manuscripts and reviews were published. The studies selected for this review extend previous knowledge and shed new and astounding knowledge on oocyte maturation. It has long been known that resumption of meiosis and progression to the metaphase II stage is intrinsic to oocyte maturation, but novel findings have revealed that specific chromatin configurations are indicative of a propensity of the oocyte to resume the meiotic process and acquire developmental competence. Recently, genetic integrity has also been characterized as a factor with important implications for oocyte maturation and quality. Changes occurring in the cytoplasmic compartment are equally fundamental. Microtubules, actin filaments and chromatin not only interact to finalize chromosome segregation, but also crucially co-operate to establish cell asymmetry. This allows polar body extrusion to be accomplished with minimal loss of cytoplasm. The cytoskeleton also orchestrates the rearrangement of organelles in preparation for fertilization. For example, during maturation the distribution of the endoplasmic reticulum undergoes major modifications guided by microtubules and microfilaments to make the oocyte more competent in the generation of intracellular Ca(2+) oscillations that are pivotal for triggering egg activation. Cumulus cells are inherent to the process of oocyte maturation, emitting regulatory signals via direct cell-to-cell contacts and paracrine factors. In addition to nurturing the oocyte with key metabolites, CCs regulate meiotic resumption and modulate the function of the oocyte cytoskeleton. CONCLUSIONS: Although the importance of oocyte maturation for the achievement of female meiosis has long been recognized, until recently much less was known of the significance of this process in relation to other fundamental developmental events. Studies on chromatin dynamics and integrity have extended our understanding of female meiosis. Concomitantly, cytoskeletal and organelle changes and the ancillary role of CCs have been better appreciated. This is expected to inspire novel concepts and advances in assisted reproduction technologies, such as the development of novel in vitro maturation systems and the identification of biomarkers of oocyte quality.

Clinical outcomes from mature oocytes derived from preovulatory and antral follicles: reflections on follicle physiology and 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.

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

Contributions of the actin cytoskeleton to the emergence of polarity during maturation in human oocytes.

In mature mammalian oocytes, cortical f-actin distribution is polarized, as evidenced by a prominent cap subtended by the metaphase II (MII) spindle. Formation of a polarized actin cap is a consequence of a complex actomyosin-driven contractile process that directs polar body extrusion. Human mature oocytes also display a network of suboolemmal actin, but so far there has been no suggestion of an actin-rich domain in the vicinity of the spindle. By high-resolution confocal microscopy, we generated semi-quantitative data of the actin cytoskeleton in human mature and immature oocytes, with the aim to better understand the characteristics and remodelling of this cytoskeletal component in the female gamete. In mature MII oocytes, the cortical domain near the spindle showed a more intense actin signal in comparison to the opposite cortical domain (177.2±59.0 versus 126.8±61.0, P<0.0001; data expressed in arbitrary units). The extent of cortical f-actin polarity was comparable between in vivo and in vitro matured oocytes. However, both the degree of polarity and relative abundance of signal were diminished with increasing maternal age. Mean intensity of cytoplasmic actin was significantly higher in oocytes matured in vitro derived from in vitro maturation (IVM) cycle, in comparison to oocytes matured in vivo or in vitro obtained from controlled ovarian stimulation cycles (35.0±8.0, 21.1±12.4 and 25.9±8.6, respectively; P=0.025). In germinal vesicle (GV)-stage oocytes obtained from both IVM and controlled ovarian stimulation cycles, cortical actin did not appear polarized, irrespective of whether the GV was located centrally or asymmetrically. These data indicate that, during maturation, cortical actin acquires a polarized distribution involving an accumulation in the domain adjacent the spindle. They also propose new questions concerning the existence of cytoplasmic actin in mature oocytes. Finally, they are suggestive of an influence of maternal age on the actin cytoskeleton.

The current challenges to efficient immature oocyte cryopreservation.

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.

Mechanistic foundations of the metaphase II spindle of human oocytes matured in vivo and in vitro.

STUDY QUESTION: Are morphometric and morphological parameters of the metaphase II (MII) spindle of human oocytes matured in vivo or in vitro predictive of chromosome alignment on the metaphase plate? SUMMARY ANSWER: Morphometric spindle parameters were very comparable between oocytes matured in vivo and in vitro and were unable to predict chromosome alignment, while a flattened shape of both poles was positively associated with chromosome displacement from the metaphase plate. WHAT IS KNOWN ALREADY: The relationship between MII spindle morphometry and chromosome alignment has only been sporadically investigated in human oocytes. The possible implications of spindle pole morphology are totally unrecognized. STUDY DESIGN, SIZE, DURATION: Morphometric and morphological analysis of the MII spindle of donated supernumerary human oocytes (N = 93) aimed at investigating possible associations between novel microtubule parameters and chromosome arrangement. PARTICIPANTS/MATERIALS, SETTING, METHODS: MII oocytes from three sources were analysed: (i) stimulated cycles matured in vivo (ivo-MII), (ii) leftover cumulus-free germinal vesicle oocytes from stimulated cycles matured in vitro (lgv-MII) and (iii) immature cumulus-cell oocyte complexes (COCs) recovered from in vitro maturation (IVM) cycles and matured in vitro (ivm-MII). Oocytes were fixed and stained for tubulin, chromatin and actin. Optical sections were collected at 0.3 µm intervals by high-performance confocal microscopy and three-dimensionally reconstructed for assignment of specific spindle and chromosomal properties. Spindle pole morphology was classified as either focused or flattened depending on whether microtubule ends were more or less convergent, respectively. Optical density measurements were generated to estimate microtubule abundance in chromosome to pole domains proximal and distal to the oolemma. MAIN RESULTS AND THE ROLE OF CHANCE: In ivo-MII oocytes, the sizes (mean ± SD) of major and minor axes were 11.8 ± 2.6 and 8.9 ± 1.7 µm, respectively, while maximum projection was 88.8 ± 29.5 µm(2). Very comparable values of these parameters were found in lgv-MII and ivm-MII oocytes. Double-focused spindles were rarely found (3.1%), unlike those with a double-flattened conformation (47.7%). Spindles with both focused and flattened poles amounted to almost half of the sample set (49.2%), but in this subgroup it was very infrequent (4.6%) to observe the flattened pole oriented towards the oolemma. Overall, differences in the relative proportions of pole morphology categories in ivo-MII, lgv-MII and ivm-MII oocytes were not statistically significant. For both the distal and proximal spindle hemidomains, optical intensity profiles were also comparable between ivo-MII, lgv-MII and ivm-MII oocytes. None of the morphometric parameters (major and minor axes, their ratio, maximum projection, distances of the metaphase plate from the poles) was associated with chromosome alignment on the metaphase plate or arrangement inside and outside the spindle. Importantly, a double-flattened outline of pole morphology was positively associated with the displacement of one or more chromosomes from the metaphase plate. Moreover, when a flattened pole was oriented towards the oolemma, a higher rate of chromosome displacement was observed. LIMITATIONS, REASONS FOR CAUTION: The findings of the study will require confirmation by further in-depth analysis and extension of the database, especially regarding the relationship between microtubule abundance and chromosome arrangement. Furthermore, considering the high number of comparisons, the observed statistical differences will require future 'ad hoc' analysis. WIDER IMPLICATIONS OF THE FINDINGS: Collectively, this work provides a robust database for future research on the human oocyte cytoskeleton, and contributes to a better definition of oocyte quality in assisted reproduction technology. Also, these data support the notion that IVM does not affect spindle morphometry and morphology. STUDY FUNDING/COMPETING INTEREST(S): Part of this work was supported by a grant awarded by the Italian Ministry of Labour, Health and Social Policies. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Oocyte in vitro maturation in normo-ovulatory women.

OBJECTIVE: To review the scientific literature concerning the application of oocyte in vitro maturation (IVM) as a treatment for normo-ovulatory women. DESIGN: Critical evaluation and comparison of the most relevant clinical studies. SETTING: Public IVF unit. PATIENT(S): Normo-ovulatory women. INTERVENTION(S): Oocyte IVM and cryopreservation. MAIN OUTCOME MEASURE(S): Maturation, fertilization, implantation, and pregnancy rates. RESULT(S): Maturation rates in vitro are suboptimal and influenced by several factors. IVM oocytes fertilize and undergo development in vitro with rates similar to in vivo matured control oocytes. In IVM cycles implantation and pregnancy rates are lower compared with controlled ovarian stimulation treatments, but accurate patient selection can improve IVM clinical outcome. CONCLUSION(S): In normal responders, IVM does not reach success rates similar to conventional ovarian stimulation regimens. However, it represents a milder approach to assisted reproduction treatment and an alternative intervention for specific conditions.

Clomiphene citrate versus high doses of gonadotropins for in vitro fertilisation in women with compromised ovarian reserve: a randomised controlled non-inferiority trial.

BACKGROUND: The aim of the present randomised controlled non-inferiority trial is to test whether in women with compromised ovarian reserve requiring in vitro fertilisation, a protocol of ovarian stimulation using exclusively clomiphene citrate performs similarly to a regimen with high doses of gonadotropins. METHODS: Women with day 3 serum FSH > 12 IU/ml on at least two occasions or previous poor response to hyper-stimulation were recruited at four Italian infertility units. Selected women were allocated to clomiphene citrate 150 mg/day from day 3 to day 7 of the cycle (n=145) or to a short protocol with GnRH agonist 0.1 mg and recombinant FSH 450 IU daily (n=146). They were randomised by means of a computer-generated list into two groups. The study was not blinded. The main outcome of the study was the delivery rate per started cycle. RESULTS: The study was interrupted after the scheduled two years of recruitment before reaching the sample size. 148 women were allocated to clomiphene citrate and 156 to the short protocol with high doses of gonadotropins; 124 and 125 participants were analysed in the groups, respectively. Women allocated to high doses of gonadotropins retrieved more oocytes and had a higher probability to perform embryo-transfer. However, the chances of success were similar. The delivery rate per started cycle in women receiving clomiphene citrate and high-dose gonadotropins was 3% (n=5) and 5% (n=7), respectively (p=0.77). The mean estimated cost per delivery in the two groups was 81,294 and 113,107 Euros, respectively. No side-effects or adverse events were observed. CONCLUSIONS: In women with compromised ovarian reserve selected for in vitro fertilisation, ovarian stimulation with clomiphene citrate or high-dose gonadotropins led to similar chances of pregnancy but the former is less expensive. TRIAL REGISTRATION: Trial registered on http://www.clinicaltrials.gov (NCT01389713).

Comparison of the obstetric and perinatal outcomes of children conceived from in vitro or in vivo matured oocytes in in vitro maturation treatments with births from conventional ICSI cycles.

STUDY QUESTION: Are the obstetric and perinatal outcomes of deliveries following in vitro maturation (IVM) cycles different from births generated from controlled ovarian stimulation (COS) cycles? SUMMARY ANSWER: The obstetric and perinatal outcomes of births from IVM cycles are comparable with those of ICSI treatments, including the incidence of major and minor abnormalities. WHAT IS KNOWN ALREADY: Only few and numerically small reports on the health of IVM children are currently available. STUDY DESIGN, SIZE AND DURATION: Retrospective cohort study involving 196 babies born from IVM cycles carried out with different priming regimens. Of these children, 79 developed from oocytes matured in vitro after 30 h of culture, while 104 originated from oocytes found mature and inseminated on the day of recovery. Thirteen babies were obtained from embryos developed from both types of oocytes. Data of these births were compared with those of 194 children born from COS ICSI cycles performed during the same period (March 2004 to December 2011). PARTICIPANTS/MATERIALS, SETTING AND METHODS: IVM cycles were done in the absence of gonadotrophin administration or with FSH and/or HCG priming. All oocytes were inseminated by microinjection. ICSI and ICSI cycles were chosen as a control group to exclude possible influences of the insemination technique. Couples in which maternal age was >39 years or affected by azoospermia were excluded to rule out major parental effects. MAIN RESULTS AND THE ROLE OF CHANCE: In single births, gestational age at delivery was comparable, but birthweight was significantly higher (P = 0.009) in children from IVM cycles (3091 ± 669 versus 3269 ± 619 g). In a separate analysis of the IVM group, comparing singleton births derived with certainty from oocytes matured in vitro (n = 71) or in vivo (n = 74), no statistically significant differences were observed in terms of birthweight (3311 ± 637 versus 3194 ± 574 g, respectively) and gestational age (38.9 ± 2.4 versus 38.4 ± 2.1 weeks, respectively). In twin births, gestational age was lower in IVM cycles, while weight at birth was comparable (ICSI, 2432 ± 540 g; IVM, 2311 ± 577 g). In single births, major and minor abnormalities were 2 (1.4%) and 6 (4.1%) in the ICSI group and 0 (0.0%) and 8 (5.2%) in the IVM category, respectively. In twin children, major and minor abnormalities were 1 (2.2%) and 2 (4.3%) in ICSI babies and 0 (0.0%) and 2 (4.6%) in IVM cycles, respectively. LIMITATIONS AND REASONS FOR CAUTION: The study is the largest conducted so far. Nevertheless, it is limited by its retrospective nature and the fact that most births of IVM treatments derived from oocytes found mature at recovery in cycles primed with HCG. A more comprehensive appraisal of the health status of IVM children will demand larger prospective studies. WIDER IMPLICATIONS OF THE FINDINGS: The study is consistent with previous reports suggesting a possible role of standard ovarian stimulation in determining a reduced birthweight in children born from COS cycles. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought to support this work. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Cleavage kinetics analysis of human embryos predicts development to blastocyst and implantation.

Cleavage kinetics of human embryos is indicative of ability to develop to blastocyst and implant. Recent advances in time-lapse microscopy have opened new and important research opportunities. In this study involving infertile couples requiring standard IVF/intracytoplasmic sperm injection treatment, zygotes were cultured by integrated embryo-culture time-lapse microscopy to analyse cleavage times from the 2- to the 8-cell stages in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after 8-cell stage, times of cleavage to 7- and 8-cell stages of embryos developing to blastocyst were shorter (56.5 ± 8.1 versus 58.8 ± 10.4h, P=0.03 and 61.0 ± 9.4 versus 65.2 ± 13.0 h, P=0.0008, respectively). In embryos developing to blastocyst, absence of blastocoele expansion on day 5 was associated with progressive cleavage delay. Implanting embryos developed to 8-cell stage in a shorter period compared with those unable to implant (54.9 ± 5.2 and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from 2- to 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on days 2 and 3 are inappropriate for accurate embryo evaluation. The speed at which human embryos cleave is known to be suggestive of their ability to develop in vitro to the blastocyst stage and implant after transfer into the uterus. Recent advances in time-lapse microscopy, which allows acquisition of images every 15-20 min, have opened new and important research opportunities. In a retrospective study involving infertile couples requiring standard IVF or intracytoplasmic sperm injection treatment, fertilized oocytes were cultured by an integrated embryo-culture time-lapse microscopy system in order to perform an analysis of cleavage times from the 2- to the 8-cell stage in relation to the ability to develop to blastocyst, expand and implant. In comparison to embryos arresting after the 8-cell stage, times of cleavage to the 7- and 8-cell stage of embryos that developed to blastocyst were significantly shorter (56.5 ± 8.1h versus 58.8 ± 10.4h and 61.0 ± 9.4h versus 65.2 ± 13.0 h, respectively). In embryos developing to the blastocyst stage, absence of blastocoele expansion on day 5 was associated with a progressive cleavage delay. Implanting embryos developed to the 8-cell stage in a shorter period compared to those unable to implant (54.9 ± 5.2h and 58.0 ± 7.2h, respectively, P=0.035). In conclusion, cleavage from the 2- to the 8-cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst, expand and implant. Conventional observation times on day 2 and 3 are appropriate for accurate embryo evaluation.

LH-receptor gene expression in human granulosa and cumulus cells from antral and preovulatory follicles.

CONTEXT: Human granulosa cells (GC) acquire LH receptor (LHR) expression during the follicular phase of the menstrual cycle. Currently, the precise follicular stage is unknown, and specific roles of LH in the follicular development are not fully understood. OBJECTIVE: Our objective was to measure LHR gene expression on GC and cumulus cells (CC) from normal human follicles with diameters form 3-20 mm. DESIGN, SETTING, AND PATIENTS: At a university hospital, GC, CC, and the corresponding follicular fluid (FF) were collected from patients undergoing fertility preservation by having one ovary frozen and patients undergoing infertility treatment. INTERVENTIONS: Cells and fluids were isolated from surgically excised ovaries or from aspirated preovulatory follicles. MAIN OUTCOME MEASURES: We evaluated gene expression of LHR, FSHR, androgen receptor (AR), aromatase (CYP19a1), and AMHR2 normalized to the GAPDH expression and associated with FF levels of anti-Mullerian hormone, inhibin-B, and steroids. RESULTS: LHR expression was maximal in GC from preovulatory follicles before ovulation induction. A majority of 150 antral follicles (3-10 mm in diameter) showed LHR expression at approximately 10% of the maximum, and LHR expression showed significant associations with FSHR, AR, CYP19a1, and AMHR2 and with FF estradiol and progesterone. Levels of FSHR continued to decline in GC as the follicular diameter increased. CONCLUSIONS: The LHR gene is expressed in GC of human antral follicles throughout the follicular phase and is significantly associated with expression of the CYP19a1 gene and with the corresponding FF concentrations of estradiol and progesterone. LH appears to affect human follicular development during most the follicular phase in normal women.