Search

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.

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.

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., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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.

Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

Objective: To assess the applicability of oocyte IVM in case of nonobstructive azoospermia (NOA)., Design: Case series., Setting: Private IVF unit., Patient(s): All male partners were affected by NOA. Twenty-one women (20 normo-ovulatory and 1 with polycystic ovary) underwent 27 IVM treatments. The outcome of controlled ovarian stimulation (COS) cycles in normo-ovulatory women whose partners were affected by NOA was assessed for comparison., Intervention(s): Spermatozoa from testicular sperm extraction (TESE) samples were retrieved and cryopreserved before treatment., Main Outcome Measure(s): Rates of fertilization, pregnancy per cycle and transfer, and implantation., Result(s): Twenty-seven IVM-TESE cycles were carried out in 21 women. Fertilization rate was 64.4%. In compliance with national legislation, no more than three oocytes were inseminated in each cycle and all developing embryos were transferred. Six single clinical pregnancies were ascertained (22.2% and 27.3% pregnancy rates per cycle and per transfer, respectively). One pregnancy ended in abortion. The others developed to term and gave rise to five healthy babies. Sixty couples underwent standard COS-TESE treatment. Rates of fertilization, clinical pregnancy per cycle, implantation, and abortion were 64.6%, 20.0%, 11.3%, and 16.7%, respectively. Eleven babies were born from ten pregnancies., Conclusion(s): Oocyte IVM may represent an option in NOA cases., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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.

This study was designed to determine if the efficiency of in-vitro maturation (IVM) in women with normal ovaries can be improved by gonadotrophin administration. 400 women were randomly allocated in four groups: group A, non-primed cycles; group B, human chorionic gonadotrophin (HCG)-primed cycles; group C, FSH-primed cycles; and group D, FSH- plus HCG-primed cycles. There were significant differences in the IVM rate among the groups. In groups where HCG was used, the overall maturation rate was higher (57.9% in group B and 77.4% in group D; 48.4% in group A and 50.8% in group C) and the percentage of total available metaphase II-stage oocytes was higher (60.4% in group B and 82.1% in group D; 48.4% in group A and 50.8% in group C). The overall clinical pregnancy rate per transfer (CPR) was 18.3% and the implantation rate (IR) was 10.6%. There was a difference in CPR among the groups: group D (29.9%) versus group A (15.3%), P = 0.023; group D versus group B (7.6%), P < 0.0001; group D versus group C (17.3%), P = 0.046. The results of this study are clearly in favour of FSH plus HCG priming. FSH priming and HCG priming alone showed no significant effects on clinical outcome.

In March 2004, a new law was introduced in Italy to regulate assisted reproduction; at present it is impossible to use more than a maximum of three oocytes per IVF cycle, nor can embryos or prezygotes (2PN cells) be selected or cryopreserved. The prohibitions introduced by the new law have, on the one hand, reduced the expectations of success of current techniques and, on the other hand, stimulated clinicians and embryologists to work on new therapeutic strategies so as to offer the highest chances of success with the lowest risks. In-vitro maturation (IVM) of oocytes fits very well with these new requirements: ovarian stimulation is avoided and the handling of spare oocytes is facilitated. The IVM protocol is an intriguing alternative to conventional IVF techniques, since it removes the side-effects of drug stimulation, especially ovarian hyperstimulation syndrome, and it also reduces the costs of the entire procedure, both in terms of 'time consumption' and 'patient/society costs for drugs'. In the authors' IVF centre the IVM technique has been used for more than a year, with significant success in terms of maturation and fertilization rates, percentage of embryo transfers, number of pregnancies and, finally, healthy babies born.

Objective: To compare the operative and postoperative course in patients undergoing laparoscopy for dermoid cyst to that observed in subjects with other types of ovarian masses and of patients undergoing laparotomy for teratomas., Study Design: Retrospective analysis. From 1994 to 1996, 49 women underwent laparoscopic cystectomy for dermoid cysts. The operative and postoperative course was compared to that of 190 patients undergoing operative laparoscopy for other adnexal masses and to that of 43 patients undergoing laparotomy for dermoid cysts from 1992 to 1996. The cysts were aspirated to reduce spillage and removed via a laparoscopic bag inserted in a 10-mm trocar. Culdotomy was never used. The abdominal cavity was abundantly flushed during the procedure and before closure., Results: Dermoid cystectomy was successfully performed laparoscopically in 47 of 49 cases. Spillage occurred in 43 cases (88%), and postoperative fever occurred in 3 (6.1%). No case of peritonitis was recorded. Significant differences between laparoscopy and laparotomy were observed in the rate of bilaterality (4% vs. 25%), spillage (88% vs. 9%) and mean hospital stay (37 vs. 83 hours). When laparoscopic excision of dermoid cysts and other masses was compared, we did not observe any significant difference in operative time or complication rates, apart from transient fever., Conclusion: Laparoscopy is safe and effective for dermoid cysts and allows shorter hospitalization than laparotomy. As observed for other benign cysts, laparoscopy should become the technique of choice for the removal of most, if not all, ovarian dermoid cysts.

18 males with beta-thalassaemia major were studied; mean age was 17.6 flAF 4.3, 8 of 18 males evaluated were prepubertal (Tanner 1), 5 were early and mid pubertal (Tanner 2-3) and 5 were full pubertal (Tanner 5). All patients (pts) underwent GnRH test and 61% showed positive response. 8 pts. were treated with hCG; 2 were azoospermic, 6 were aspermic. The starting dose was 1000 IU twice weekly; dose adjustments were made basing on testosterone levels and clinical features. Clinical examination, testosterone assays, semen analysis were performed every 3 months during the treatment period. Therapy was started 18-48 months ago and it is still in progress. Within 12 months of treatment 4 pts progressed to Tanner stage 4, 3 pts to Tanner stage 5, 1 showed no response. Semen analysis showed an improvement in 6 pts: after 12 months of therapy 1 pts was aspermic, 5 were azoospermic, 1 was oligozoospermic. Our data showed that hCG administration is effective in inducing and maintaining secondary sexual character.

The aim of the study is to evaluate the results of tests performed in "infertile" couples that have obtained spontaneous pregnancy. 953 couples were studied from 1986 to 1991; 108 spontaneous pregnancies occurred (11.1%). All patients were evaluated according to standard procedures recommended by literature. "Female factor" as cause of infertility was present in 41.5%; "cervical factor" was present in 19.4%; "male factor" was present in 48.1% while in 14.8% of the couples evaluated no pathology was showed. In this series, spontaneous pregnancy occurred within 4 year of infertility in more than 90%; no significant difference was present between primary and secondary infertility. A considerable incidence of pathologies was observed in couples that obtained spontaneous pregnancy. Our data suggest that common investigation for infertility may have not a real prognostic value.