Cryopreservation Of Immature Oocytes Research Articles

P-443 Evaluation of the effects of a semi-automated vitrification performed before or after in vitro maturation (IVM) on the kinetic of oocyte maturation and chromosome segregation

Abstract Study question What are the optimal vitrification method (semi-automated vs. manual) and oocyte stage for in vitro -matured oocyte cryopreservation using meiosis kinetics and chromosome segregation as readouts? Summary answer The semi-automated vitrification method does not impact oocyte nuclear maturation quality compared with the manual method. Immature oocyte cryopreservation should be performed after IVM. What is known already Fertility preservation using oocyte vitrification should be performed before oncological treatments. The reference protocol consists in collecting mature oocytes after ovarian stimulation. Nevertheless, ovarian stimulation sometimes yields immature oocytes or cannot be performed (e.g., emergency oncological treatment). An IVM step is therefore required but it is not clearly demonstrated whether IVM should be performed before or after vitrification. Oocyte vitrification is usually performed with manual methods. A semi-automated vitrification device (Gavi®, Genea Biomedx) showing high performances for embryo was recently released. To our knowledge, no study has analysed its efficiency on oocyte vitrification. Study design, size, duration 200 immature oocytes collected from ICSI cycles from January 2020 will be used. Oocytes will be divided in five groups (40 oocytes/group): freshly matured oocytes (group 1 control), oocytes vitrified after IVM by a manual technique (group 2a) or by Gavi® (group 2b) and oocytes vitrified prior IVM (groups 3a and 3b). We assess oocyte nuclear maturation quality by evaluating IVM kinetics by time-lapse (Geri®) and the accuracy of homologous chromosomes segregation by CGH array. Participants/materials, setting, methods Since January 2020, 124 out of 200 immature oocytes have been included for this study. These oocytes provide from women under 37 years old without ovulatory disorder after signing an informed consent. The kinetics of meiotic resumption (germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) timings), is determined by time-lapse technology (Geri®, Genea Biomedx). The accuracy of the homologous chromosome segregation during the first meiotic division will be assessed by CGH-Array. Main results and the role of chance The clinico-biological characteristics (age, BMI, smoking and total FSH dose) are comparable between the five groups (p > 0.05). No significant difference in post-thawing oocyte survival rate is observed between the two vitrification methods (semi-automated 58% vs. manual 64%). A significant difference in the overall oocyte survival rate is observed according to the stage of vitrification with a significantly higher survival rate if oocytes are vitrified at the mature stage (93% (2a+2b) vs. 61% (3a+3b), p = 0.02). The IVM rate is significantly higher if oocytes are matured freshly (86% in group 1 (fresh IVM, n = 7) and 93% in group 2a+2b (IVM before vitrification, n = 27)) compared to post vitrification (71% in group 3a+3b, n = 28), p = 0.03. The vitrification technique does not seem to impact IVM rate since it reached 64% (group 3a, n = 11) and 76% (group 3b, n = 16) (p = 0.4). GVBD and PBE timings are not significantly different between the 5 groups, suggesting that neither the oocyte stage of vitrification nor the vitrification technique affects maturation kinetics. Similarly, our preliminary results of polar bodies and oocytes chromosomal profiles assessed by CGH-Array demonstrate a similar rate of aneuploidy (monosomy or trisomy) between the groups. Limitations, reasons for caution Our preliminary results of CGH array should be confirmed with the analysis of a larger number of IVM oocytes. Wider implications of the findings Vitrification of immature oocytes should be performed by semi-automated or manual methods after IVM. Tour knowledge, this is the first study comparing the efficiency of both semi-automated and manual vitrification methods on immature and in vitro-matured oocytes. Trial registration number NCT03680937

Cryopreservation of Immature Oocytes at Germinal Vesicle Stage. When Gamete Maturation Performance Seems to Be Most Appropriate?

Fertility preservation is among the priorities in reproductive medicine. However, the cancer patients and women with various functional ovarian disorders, wishing to preserve future reproductive potential may have some contraindications or no possibilities to cryopreserve mature oocytes and ovarian tissue. Therefore, the development of techniques for immature oocyte cryopreservation is considered an alternative strategy. Here, we have evaluated the survival, maturation, fertilization and embryo development rates of immature oocytes (Germinal vesicle (GV) stage – group 1) after cryopreservation and in vitro matured (IVM) ones (group 2) prior to cryopreservation, compared with in vivo matured metaphase-II (MII) oocytes (group 3). Survival rates were 97.6, 96.2 and 98.2 % for groups 1–3, respectively. The maturation rate of GV oocytes in group 1 was significantly lower than in group 2 and made 52.0 and 73.2%, respectively. The highest fertilization rate was revealed in group 3, and the lowest one was in group 1. The groups 1–3 showed the same tendency for further embryo development, i. e. the blastulation rates were 20.0, 38.5 and 56.9%, respectively. Thus, the survival rate of cryopreserved oocytes did not depend on their maturity rate. However, the IVM oocytes displayed lower fertilization and blastulation rates, than the in vivo matured ones. It was found that oocytes IVM should be performed prior to cryopreservation, because it ensured higher rates of maturation, fertilization and embryo development in vitro.

FERTILITY PRESERVATION IN YOUNG WOMEN WITH EARLY-STAGE BREAST CANCER.

SUMMARYAlthough breast cancer (BC) occurs more often in older women, it is the most commonly diagnosed malignancy in women of childbearing age. Owing to the overall advancement of modern medicine and the growing global trend of delaying childbirth until later age, we find ever more younger women diagnosed and treated for BC who have not yet completed their family. Therefore, fertility preservation has emerged as a very important quality of life issue for young BC survivors. This paper reviews currently available options for fertility preservation in young women with early-stage BC and highlights the importance of a multidisciplinary approach to fertility preservation as a very important quality of life issue for young BC survivors. Pregnancy after BC treatment is considered not to be associated with an increased risk of BC recurrence; therefore, it should not be discouraged for those women who want to achieve pregnancy after oncologic treatment. Currently, it is recommended to delay pregnancy for at least 2 years after BC diagnosis, when the risk of recurrence is highest. However, BC patients of reproductive age should be informed about the potential negative effects of oncologic therapy on fertility, as well as on the fertility preservation options available, and if interested in fertility preservation, they should be promptly referred to a reproductive specialist. Early referral to a reproductive specialist is an important factor that increases the likelihood of successful fertility preservation. Embryo and mature oocyte cryopreservation are currently the only established fertility preservation methods but they require ovarian stimulation (OS), which delays initiation of chemotherapy for at least 2 weeks. Controlled OS does not seem to increase the risk of BC recurrence. Other fertility preservation methods (ovarian tissue cryopreservation, cryopreservation of immature oocytes and ovarian suppression with gonadotropin-releasing hormone agonists) do not require OS but are still considered to be experimental techniques for fertility preservation.

Chapter 3 Current Challenges in Immature Oocyte Cryopreservation.

Current freezing technology, especially the vitrification method, has markedly improved oocyte survival rate after warming, and the pregnancy rate is comparable to that achieved with fresh oocytes. However, most groups report using oocytes matured in vivo for vitrification. Although immature oocytes can be vitrified successfully, clinical outcomes do not reach that of vitrification of matured oocytes. The current literature suggests that oocytes should be vitrified at mature metaphase II (M-II) stage following IVM rather than at the immature germinal vesicle (GV) stage, because the potential for oocyte maturation is reduced when vitrification is performed on immature oocytes at the GV stage.

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.

Production of Live Offspring from Vitrified-Warmed Oocytes Collected at Metaphase I Stage.

Vitrification of matured oocytes is widely adopted in human clinics and animal research laboratories. Cryopreservation of immature oocytes, particularly those at metaphase I (MI), remains a challenge. In the present work, mouse MI oocytes denuded of cumulus cells were vitrified and warmed (V/W) either prior to (V/W-BEFORE-IVM, n = 562) or after (V/W-AFTER-IVM, n = 664) in vitro maturation (IVM). Derivative metaphase II (MII) oocytes were then used for intracytoplasmic sperm injection (ICSI). In the control groups, in vivo matured MII oocytes were used freshly (FRESH-MII, n = 517) or after V/W (MII-V/W, n = 617). In vitro and in vivo developmental competencies were compared among groups. Satisfactory blastocyst rates were achieved in V/W-BEFORE-IVM (27.5%) and V/W-AFTER-IVM (32.4%) groups, albeit as expected still lower than those from fresh-MII (56.1%) or MII-V/W (45.6%) oocytes. Similarly, the term development rates from V/W-BEFORE-IVM and V/W-AFTER-IVM were 12.4% and 16.7% respectively, acceptable but lower than those of the fresh-MII (41.2%) and MII-V/W (23.3%) groups. These data demonstrate that oocytes collected at MI stage are amenable to V/W, which can be performed before or after IVM with acceptable development rates including production of healthy pups. These findings provide useful knowledge to researchers and clinical practitioners for preservation and use of the otherwise discarded MI oocytes.

Influence of various cryoprotectors on survival of sheep’s ovarian tissuewith freezing in vapors of liquid nitrogen

Biological diversity is the key to maintaining life. Therefore preservation of biodiversity is important for sustaining a healthy Earth, but it also is immensely valuable tothe health and lifestyle of human society. Today new methods of auxiliary reproductivetechnology allow using them not only for preservation of reproductive potential of theperson, and also for preservation a biodiversity of wild and endangered species of farmanimals. Captive breeding programs, genetic resource banks and artificial reproductivetechniques have been suggested as important tools for conservation. Today, biotechnological methods, such as artificial insemination and transplantation of embryos are welldeveloped and are used in programs of animal husbandry and preservation. Receivingoocytes and embryos requires stimulation of ovaries on what some time depending ona species of animals usually is required. These methods are not acceptable in case ofemergency, and the in vivo received oocytes in smaller quantity do not guarantee themaximum storage of genetic material. Therefore an alternative method of preservation ofgenetic material is the cryopreservation of immature oocytes in the primordial follicleswhich are located in ovary cortex. The procedure of ovarian tissue cryopreservation permits conservation of hundreds of immature oocytes kept within the protective environment of the original ovarian tissue. An important advantage of this technique that thehormonal stimulation is not required in this case. Additionally, because the primordialfollicles are small and have a simple structure, they are much more tolerant to manipulation and to the freeze-thaw procedure compared with the large growing follicles. A keyelement of a good cryopreservation to cell survival is the physiochemical relationshipof heat and water transport between the intra- and extracellular environment. The art ofcryobiology involves the addition of one or more cryoprotectants which generally reduceboth the eutectic and freezing points. Therefore the purpose of this work is identificationof an optimum method of a cryopreservation of sheep‘s ovarian tissue using of variouscryoprotectors. In the present study, the cryopreservation of indigenous Chui population sheep’s ovarian tissue was conducted in vapors of liquid nitrogen at distance of 5cm from surface within 20 minutes with use of various cryoprotectors: 1,5 M dimethylsulfoxide (DMSO), 1,5 M propylene glycol (PG), 1,5 M ethylene glycol (EG) and 1,5 Mglycerol (HL). The analysis of comparative histology studying shows that use 1,5 M PGand 1,5 M DMSO has more effective effect on viability of ovarian follicles, than use 1,5M EG and 1,5 M HL.Key words: dimethyl sulfoxide, ethylene glycol, freezing, follicles, glycerol, ovariantissue, propylene glycol.

41 DEVELOPMENTAL POTENTIAL OF BUFFALO OOCYTES VITRIFIED AT THE GERMINAL VESICLE STAGE: EFFECTS OF DIFFERENT CRYOPROTECTANT COMBINATIONS AND CRYODEVICES

The cryopreservation of immature oocytes would generate a readily available, nonseasonal source of female gametes for both research and reproduction. In domestic animals, the most promising results in the field of oocyte cryopreservation have been reported in cattle, and a few experiments have been conducted on buffalo. The aim of the present study was to compare the effects of different cryoprotectant combinations and different cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle stage. Cumulus-oocyte complexes obtained at slaughter from mature buffalos were vitrified by using either straw or open pulled-straw or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol or 20% EG + 20% dimethylsulfoxide (DMSO). Following vitrification and warming, viable oocytes were matured in vitro for 22 h. Matured oocytes were either evaluated for nuclear maturation or fertilized and cultured in vitro for 7 days. Recovery rate was significantly higher (P &lt; 0.05) in the oocytes vitrified by straw in 20% EG + 20% glycerol (92.6%) as compared with the other groups. Percentages of viable oocytes were significantly higher (P &lt; 0.05) in the oocytes vitrified in 20% EG + 20% DMSO using SSV (95.7%) than those in the other groups (from 80 to 88.0%). Among the vitrified groups, the highest maturation rate was achieved in SSV with 20% EG + 20% DMSO group (56.7%). This value was comparable with those in the control group (62.1%). After IVF and embryo culture, the highest cleavage and blastocyst rates were obtained in SSV with 20% EG + 20% DMSO group (35.7 and 21.4%, respectively), and these values were nearly similar to those in the control group (38.7 and 25.8%, respectively). Vitrification of oocytes by straw or open pulled-straw resulted in significantly lower (P &lt; 0.05) blastocyst rates (2.6 and 11.5%) as compared with the control. In conclusion, buffalo oocytes vitrified at the germinal vesicle stage can be matured, fertilized, and developed in vitro and produce high frequencies of blastocyst embryos. Solid surface vitrification may be superior to straw and open pulled-straw in vitrification of immature buffalo oocytes because this technique results in higher survival and embryo development rates.

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

Female fertility: is it safe to "freeze?".

Objective:To evaluate the safety and risk of cryopreservation in female fertility preservation.Data sources:The data analyzed in this review were the English articles from 1980 to 2013 from journal databases, primarily PubMed and Google scholar. The criteria used in the literature search show as following: (1) human; embryo; cryopreservation/freezing/vitrification, (2) human; oocyte/immature oocyte; cryopreservation/ freezing/vitrification, (3) human; ovarian tissue transplantation; cryopreservation/freezing/vitrification, (4) human; aneuploidy/DNA damage/epigenetic; cryopreservation/freezing/vitrification, and (5) human; fertility preservation; maternal age.Study selection:The risk ratios based on survival rate, maturation rate, fertilization rate, cleavage rate, implantation rate, pregnancy rate, and clinical risk rate were acquired from relevant meta-analysis studies. These studies included randomized controlled trials or studies with one of the primary outcome measures covering cryopreservation of human mature oocytes, embryos, and ovarian tissues within the last 7 years (from 2006 to 2013, since the pregnancy rates of oocyte vitrification were significantly increased due to the improved techniques). The data involving immature oocyte cryopreservation obtained from individual studies was also reviewed by the authors.Results:Vitrifications of mature oocytes and embryos obtained better clinical outcomes and did not increase the risks of DNA damage, spindle configuration, embryonic aneuploidy, and genomic imprinting as compared with fresh and slow-freezing procedures, respectively.Conclusions:Both embryo and oocyte vitrifications are safe applications in female fertility preservation.

Cryopreservation of immature porcine oocytes by Cryotop

In our paper, porcine oocytes at GV stage were vitrified in cryotops, thawed, and subsequently in vitro matured. Cumulus-oocyte complexes (COCs) were collected from follicles of 4-6 mm in diameter. Some of them were fixed right after collection to evaluate development status before vitrication and/or IVM (group 1). The others were subjected to: normal IVM (group 2); treatment with vitrification media-(group 3); vitrified by cryotops (group 4); centrifugation before vitrified by cryotops (group 5). The results showed that all oocytes in group 1 were at the GV stage. The percentages of cumulus expansion, oocytes showing normal morphology, and MII oocytes in groups 2 and 3 showed no significant difference (100, 100 and 84% vs. 100, 100 and 82%, respectively). Those of group 4 were lower (79%, 68% and 57%, respectively). Group 5 had lowest rates (13%, 23% and 7%, respectively). Similar results of were obtained when oocytes of groups were treated with fluorescein diacetate (FDA) và propidium iodide (PI) for live/dead cell evalutation. These results indicate that vitrification by cryotops can be an effective method for cryopreservation of immature oocytes in pigs before IVM and centrifugation is not needed for successful vitrification.

223 CRYOPRESERVATION OF IMMATURE BOVINE CUMULUS–OOCYTE COMPLEXES BY SLOW RATE FREEZING AND VITRIFICATION

Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.

Fertility preservation in young patients' with cancer.

Preservation of fertility is an important issue in the management of young cancer patients. Though embryo cryostorage is a well-established procedure, it can only be availed by couples. Recent studies have indicated increasing success rates with mature and immature oocyte cryopreservation. Cryostorage induces injuries on the human oocytes which can be minimized by slow freezing and vitrification. Selection of candiidates is crucial so that the most suitable technique can be offered without any delay in initiation of cancer therapy. Factors affecting suitability are age of patient, assessment of ovarian reserve, hormonal status and type and stage of neoplastic disease. Encouraging results have been obtained with oocyte in vitro maturation (IVM) followed by vitrification for cryostorage. Data on the use of vitrified eggs in routine in vitro fertilization (IVF) show that pregnancy rates can be comparable to those achieved with fresh oocytes.

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.

Fertility preservation in young breast cancer patients: addressing individual patient needs

SUMMARY In female patients, infertility may be regarded as an essential loss of femininity and infertility after cancer, and is known to be associated with psychosocial distress. Fertility preservation is thus an important issue for young women diagnosed with breast cancer. Nevertheless, breast cancer patients have traditionally not been offered the most well-established methods for fertility preservation, such as embryo and oocyte freezing, as a rise in estradiol during standard stimulation protocols has been regarded as potentially deleterious in these patients. Alternative protocols for stimulation, such as those using tamoxifen and aromatase inhibitors, have therefore been developed. Protocols including aromatase inhibitors have been demonstrated to be more effective than those using tamoxifen in terms of the number of oocytes and embryos obtained, while maintaining estradiol levels within the normal range of a menstrual cycle. Cryopreservation of immature oocytes and ovarian cortical tissue are methods that are still under development. Reports of children born after application of these experimental methods are currently increasing and these methods are thus gaining a place within the options for fertility preservation in breast cancer patients.

Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop

The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus-oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P<0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

Live birth after in vitro maturation and vitrification of immature oocytes retrieved from conventional IVF cycle: a case report

Oocyte cryopreservation is gaining increased recognition as a viable alternative to embryo freezing since this technique satisfied several concerns about ethical, religious and social constraints that are associated with the creation and storage of embryos, and allowed donated oocytes to be used following quarantine in a more cost-effective manner and offered women an ideal modality to not only preserve their fertility but also their reproductive autonomy [1]. Despite this, oocyte cryopreservation is technically challenging and it has been more than two decades since the first successful report of oocyte cryopreservation using the slow-freezing method [2]. Although additional reports of successful oocyte freezing and normal pregnancies followed, the slow-freezing method did not become a routine IVF procedure because of its inefficiency and inconsistency [3]. In contrast, vitrification has been reported more recently as an alternative cryopreservation method that is characterized by simplicity, low cost, and high efficiency for cryopreserving both oocytes and embryos. Oocyte vitrification offers increased success rates in comparison to the slow-freezing method. There are now over 1500 live births resulting from IVF oocyte vitrification reported in the literature [4–6]. In addition, several IVF centers in the world have reported pregnancy rates similar to those of standard fresh IVF treatments [7, 8]. However, very few live births have been reported after cryopreservation of immature oocytes either before in vitro maturation (IVM) [9] or after IVM [10, 11]. The first live birth after slow-freezing of the immature oocytes retrieved from conventional IVF cycles was reported in 1998 [9]. Recently, 4 pregnancies with live birth from immature oocytes retrieved in a natural menstrual cycle, followed by IVM and cryopreservation of the oocytes by vitrification have been reported [10, 11]. However, there has been no report whether the mature oocytes produced by IVM of immature oocytes obtained from conventional IVF cycles can be vitrified and develop into viable embryos. In this case report, we present a pregnancy resulting from transfer of vitrified IVM oocytes generated from immature oocytes of a conventional IVF cycle. To the best of our knowledge, this is the first report of a live birth from vitrification of oocytes produced by IVM of immature oocytes derived from the controlled ovarian hyperstimulation (COH) cycles of a patient in the oocyte donation program.

In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage

Cryopreservation of immature oocytes at germinal vesicle (GV) stage would provide a readily available source of oocytes for use in research and allow experiments to be performed irrespective of seasonality or other constraints. This study was designed to evaluate the recovery, viability, maturation status, fertilization events and subsequent development of ovine oocytes vitrified at GV stage using solid surface vitrification (SSV). Cumulus oocyte complexes (COCs) obtained from mature ewes were randomly divided into three groups (1) SSV (oocytes were vitrified using SSV), (2) EXP (oocytes were exposed to vitrification and warming solutions without vitrification) or (3) Untreated (control). Following vitrification and warming, viable oocytes were matured in vitro for 24h. After that, nuclear maturation was evaluated using orcein staining. Matured oocytes were fertilized and cultured in vitro for 7days. Following SSV, 75.7% 143/189 oocytes were recovered. Of those oocytes recovered 74.8%, 107/143 were morphologically normal (viable). Frequencies of in vitro maturation were significantly (P<0.01) decreased in SSV and EXP groups as compared to control. In vitro fertilization rates were significantly (P<0.01) decreased in SSV (39.3%) group as compared to EXP (56.4%) and control (64.7%) groups. Cleavage at 48h post insemination (pi) and development to the blastocyst stage on day 7 pi were significantly (P<0.001) decreased in SSV oocytes as compared to EXP and control groups. In conclusion, immature ovine oocytes vitrified using SSV as a simple and rapid procedure can survive and subsequently be matured, fertilized and cultured in vitro up to the blastocyst stage, although the frequency of development is low.

The use of immature oocytes in the fertility preservation of cancer patients: current promises and challenges

Improved oncological treatments permit increased survival rates, although cancer patients remain at risk of losing ovarian function. An attractive option for fertility preservation includes the use of immature oocytes, a strategy which can occur on a rapid timeline and without hormonal stimulation. As a result, cancer therapy can proceed promptly even in patients with hormone-sensitive tumors. Following retrieval, immature oocytes can be cryopreserved at either the immature germinal vesicle or the mature metaphase-II stage, i.e. either before or after in vitro maturation (IVM). We present a critical review of previous human studies on the cryopreservation of immature oocytes. Evaluations include in vitro developmental competence upon thawing/warming, or organization of the spindle and chromosomes. Reported successes vary, perhaps in relation to the source of the oocytes and protocols for cryopreservation and IVM. Weaknesses exist with the experimental designs implemented to date, so caution must be exercised before considering the use of immature oocytes to be a safe and reliable practice in the fertility preservation of cancer patients. To date, results indicate that with current protocols, it may be best to cryopreserve immature oocytes after IVM at the metaphase-II stage. Nonetheless, efficacy remains very low. Future efforts should tailor and optimize not only cryo-preservation, but also IVM protocols for use in either germinal vesicle or metaphase-II oocytes, together with a comprehensive assessment of oocyte function and developmental competence to term. Despite current challenges, the burgeoning field of immature oocyte cryopreservation constitutes a promising option for cancer patients with impaired ovarian function.

Human Oocyte Cryopreservation - An Emerging ART Technique: Are We Heading in the Right Direction?

Oocyte cryopreservation is a promising adjunct to human assisted reproductive technology. Slow rate freezing has been the cryopreservation standard for storage of sperm, embryos and, subsequently, oocytes. However, earlier concerns regarding damage to the meiotic spindle, loss of cortical granules and the low success rates as compared to the relative success of embryo cryopreservation caused little interest until the 1990s. Interest increased when many studies indicated that acceptable oocyte survival, in vitro fertilization, normal embryos and adequate blastocyst development can be achieved with oocyte cryopreservation. Recently introduced oocyte vitrification techniques are proving to be more efficient. Survival rates are close to 100% and developmental rates are similar to those achieved with fresh oocytes. This efficiency opens the way to the widespread application of the technique in various medical, legal, and social situations, even to replace embryo cryopreservation with the oocyte cryopreservation. Oocyte vitrification has dominated slow freezing to such an extent that it may soon become the exclusive cryopreservation choice, especially considering that potential disease transmission problems commonly associated with vitrification due to direct exposure of oocytes to liquid nitrogen can be eliminated by using the proper techniques and devices. Furthermore, cryopreservation of immature oocytes, ovarian follicles, ovarian tissue and whole ovary are other emerging technologies. Oocyte cryopreservation has tremendous opportunity for preserving fertility in cancer patients, for those who may not have sperm following oocyte retrieval and for those women who wish to delay their motherhood. The purpose of this article is to review the history of oocyte cryopreservation, its applications, current cryopreservation techniques and future trends for fertility cryopreservation, to determine if oocyte cryopreservation is proceeding in the right direction.