Resumption of meiosis-I tissue to enucleated preovulatory oocytes: a preliminary report

Purpose : Ovarian tissue banking may be the best strategy to preserve female fertility. But optimal method to obtain viable mature oocytes remains challenging. In order to bypass the long in vitro oocyte growth period, we developed this study to test whether reconstruction of thawed primordial oocytes with enucleated preovulatory germinal vesicle (GV) oocytes could induce dictyate nuclei to undergo chromosomal condensation and meiotic maturation. Methods : Isolated primordial oocytes from thawed mouse ovarian tissue were reconstructed with enucleated GV oocytes. After electrofusion and in vitro maturation, the reconstituted oocytes were assessed for first polar body extrusion, cytoskeleton configuration, and chromosome abnormalities. Results : Primordial oocytes from thawed ovarian tissue showed a high survival rate. Following transfer and electrofusion, they could be fused with enucleated GV oocytes (35.6%, 36/101) and extruded a first polar body (52.8%, 19/36). These mature oocytes showed a normal spindle configuration and chromosome number. Conclusions : We successfully established a mouse cell model to prove that omitting the whole growth and maturation period by transfer of primordial oocytes to developmentally older enucleated oocytes would bypass the long growth period required to the preovulatory stage. Polar body extrusion could also ensue after in vitro growth. This study provided an alternative approach for future investigations in oocyte maturation.     

Successful delivery following cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome-like disease: a case report

Purpose : To estimate frozen zygotes, which developed from in vitro matured oocytes retrieved from polycystic ovarian syndrome-like disease. Methods : Oocyte retrieval was performed on Day 15 following withdrawal bleeding. The oocytes were incubated for 24 h in TCM-199 maturation medium supplemented with follicle fluid, E2, FSH, and hCG. Results : A total of 12 immature oocytes were collected. Seven of the 12 oocytes (58.3%) developed to the metaphase-II stage, and subsequently, all seven fertilized oocytes were frozen at the pronuclear stage. The remaining five oocytes failed to develop to the metaphase-II stage after an additional 24 h of incubation. Three of seven cryopreserved oocytes were thawed and developed to 2–8-cell cleaved stage embryos. The first pregnancy failed. However, the second frozen–thawed embryo transfer resulted in the delivery of healthy twins. Conclusions : Successful delivery using frozen zygotes from an anovulatory woman with polycystic ovarian syndrome-like disease.     

冷冻保存人类未成熟卵母细胞对其发育潜能的影响

目的 评价冷冻保存人类未成熟卵母细胞对其发育潜能的影响。方法 收集常规胞质内单精子显微注射-胚胎移植（ICSI-ET）中不成熟的卵母细胞168个,根据成熟程度分为生发小泡期（GV）卵母细胞103个和第1次减数分裂中期（MI）卵母细胞65个,再将各期卵母细胞分别分为冷冻组和对照组,冷冻组细胞经慢速冷冻-快速融解（慢冻-速融）后在体外培养成熟,对照组直接进行体外培养成熟。最后进行免疫荧光染色并观察。结果 GV冷冻组和GV对照组卵母细胞之间体外成熟率（分别为69．1％、74．3％）比较,差异无统计学意义（P〉0．05）;GV冷冻组和GV对照组卵母细胞之间纺锤体形态正常率（分别为28．9％、53．9％）和染色体形态正常率（分别为23．7％、50．0％）比较,差异有统计学意义（P〈0．05）;MI冷冻组和MI对照组卵母细胞之间体外成熟率（分别为68．6％、88．0％）比较,差异无统计学意义（P〉0．05）。MI冷冻组和MI对照组卵母细胞之间纺锤体形态正常率（分别为20．8％、54．6％）和染色体形态正常率（分别为25．0％、63．6％）比较,差异有统计学意义（P〈0．05）;GV冷冻组和MI冷冻组之间各项结果比较,差异均无统计学意义（P〉0．05）。结论 应用常规慢冻．速融方案并不能很好地保存未成熟卵母细胞的体外成熟后的纺锤体形成能力,未成熟的卵母细胞的发育潜能从而受损。     

Recovery and Maturation of Immature Oocytes in Patients at Risk for Ovarian Hyperstimulation Syndrome

Purpose: Our purpose was to examine the rate of immature oocyte recovery and their potential for in vitro maturation from canceled human menopausal gonadotropin cycles due to the risk of having ovarian hyperstimulation syndrome develop. Methods: Patients underwent ultrasound-guided immature oocyte pickup. The number of oocytes recovered from these patients was recorded, and then cultured in vitro. Cumulus expansion and the stage of nuclear maturation were observed after 24 and 48 hr, respectively. Results: Seventeen patients underwent 20 immature oocyte recoveries. A total of 162 oocytes (8.1 oocytes/patient) was obtained. All of the oocytes were enclosed in dense layers of cumulus cells. Among them, 78.4% showed cumulus expansion after 24 hr and 66% completed meiotic maturation to metaphase II after 48 hr in culture. There was only one immature oocyte pickup in which no oocytes were recovered (95% recovery rate). None of the patients had ovarian hyperstimulation syndrome develop. Conclusions: Immature oocytes can be recovered from canceled human menopausal gonadotmpin cycles in patients who are at potential risk for severe hyperstimulation syndrome. These oocytes can be matured in vitro and can be used for clinical and research purposes as well.     

Slow and ultrarapid freezing of fully grown germinal vesicle-stage mouse oocytes: Optimization of survival rate outweighed by defective blastocyst formation

Purpose The cryopreservation of mature metaphase II-stage mouse oocytes is associated with decreased fertilizability, spindle damage, and increased polyploidy. Therefore, we investigated the outcome of cryopreservation of immature germinal vesicle-stage mouse oocytes.Methods Oocytes were punctured from Graafian follicles in primed F₁ hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing protocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both agents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each group of frozen oocytes was compared with both unfrozen germinal vesicle-stage oocytes and metaphase II-stage oocytes.Results An optimal cryosurvival rate of 78% was reached after ultrarapid freezing with 3 Mdimethyl sulfoxide followed by one-step dilution, but a decreased rate of twocell formation was observed. Freezing with a combination of dimethyl sulfoxide and 1,2-propanediol did not improve this fertilization-decreasing effect. Very low cryosurvival rates after freezing with 1,2-propanediol indicated its inappropriateness for ultrarapid freezing of immature oocytes. The rates of in vitro maturation were equivalent for frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, as a general characteristic for both slow and ultrarapid freezing of fully grown germinal vesicle-stage oocytes, that the in vitro development up to the blastocyst stage is inhibited despite full nuclear maturation. Conclusion We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmental capacity after fertilization. The rate of in vitro nuclear maturation did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.     

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. Methods: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. Results: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. Conclusions: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.     

Successful birth following transfer of frozen–thawed embryos produced from in-vitro matured oocytes

It is well established that ovarian hyperstimulation syndrome (OHSS) is more frequent in patients with polycystic ovarian syndrome. In-vitro maturation (IVM) of immature oocytes presents a potential alternative for the fertility treatment and prevention of OHSS for these patients. This report describes the case of a 26-year old woman with a successful pregnancy and delivery following the transfer of frozen–thawed embryos derived from in-vitro matured oocytes. She had three failed cycles of ovarian stimulation (using low-dose step-up gonadotrophin protocol) with or without intrauterine insemination cycles, an ovulation-induction cycle with luteal long protocol, two fresh IVM cycle and one frozen–thawed IVM cycle. During the IVF cycle, she developed moderate OHSS and required hospitalization for 3 weeks. Following four unsuccessful IVF or IVM cycles, 15 months after the last cryopreservation, six fertilized oocytes were thawed for a scheduled embryo transfer. Following thawing, four fertilized oocytes survived and cleaved. Four frozen–thawed embryos were transferred. Six weeks after embryo transfer an ongoing intrauterine single pregnancy with fetal heartbeat was confirmed by transvaginal ultrasound. An uneventful pregnancy and delivery via Caesarean section at 39 weeks resulted in the birth of a normal healthy infant.     

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。     

Meiotic spindle visualization in living human oocytes

A computer-assisted polarization microscopy system (polscope) has made it possible to analyse the meiotic spindle of oocytes subjected to intracytoplasmic sperm injection (ICSI) without affecting their viability. It has been shown that the presence of a detectable birefringent meiotic spindle inside the oocyte cytoplasm of human metaphase II (MII) prepared for ICSI is an indicator of oocyte quality, such as fertilization and developmental ability. Meiotic spindle imaging has also shown that this structure, when detectable, is not always aligned with the first polar body (PB1) in fresh MII oocytes. The relationship between the degree of meiotic spindle deviation from the PB1 location and ICSI outcomes is discussed in this paper. When the meiotic spindle of in-vitro matured oocytes is analysed, it is always found to be aligned with the PB1, suggesting that the misalignment observed in the oocytes matured in vivo results from the PB1 displacement during the manipulations for the cumulus and corona removal. Furthermore, polscope analysis of meiotic spindle changes in living MII oocytes subjected to freezing and thawing procedures has shown that the current techniques of oocyte cryopreservation cause meiotic spindle destruction. The polscope system may assist in the selection of fresh and thawed oocytes for ICSI.     

Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy

OBJECTIVE: To investigate the efficacy of the PolScope on imaging the spindle morphology in oocytes at the metaphase II stage before vitrification and after thawing. DESIGN: In vitro study. SETTING: University infertility clinic and academic research laboratory. INTERVENTION(S): Oocytes at the metaphase II stage that were obtained from superovulating mice were vitrified and then thawed. MAIN OUTCOME MEASURE(S): Morphological features of the spindle in oocytes were evaluated by both the PolScope and immunofluorescent staining. RESULT(S): Using the PolScope, the morphological features of the spindle of intact thawed oocytes were undetected by 3 hours of thawing in only 25% of cases. Most of the spindle images were recognized during the first hour of observation. Additionally, the statistical analysis of agreement of spindle morphology by both the PolScope and fluorescent staining showed a weighted Kappa value of 0.70, indicating good agreement. Oocytes with good spindle morphology verified by the PolScope before vitrification had a higher survival rate of intact oocytes after thawing compared with those with poor or undetected spindle images. CONCLUSION(S): The morphological features of the spindle in oocytes evaluated by the PolScope before freezing and after thawing are significantly correlated with those assessed by immunofluorescent staining after fixation. With the assistance of the PolScope, thawed oocytes with good spindle morphology can be verified and selected for further manipulation without fixation and staining.     

Pregnancy and delivery after in vitro maturation of naked ICSI GV oocytes with GH and transfer of a frozen thawed blastocyst: case report

Purpose : To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. Method : All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37°C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. Results : The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. Conclusion : In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.     

Letters to the editor

Objective: To describe the birth achieved from frozen embryos after intracytoplasmic sperm injection (ICSI) of donor sperm into vitrified oocytes.

Patient: A 25-year-old woman whose husband was azoospermic undergoing IVF therapy.

Methods: Oocytes collected after ovarian stimulation were vitrified, thawed, and fertilized by frozen donor sperm.

Results: Nineteen oocytes were vitrified and all survived after thawing. Thirteen of the 19 oocytes that underwent ICSI with donors sperm were successfully fertilized. Twelve embryos were cryopreserved again by conventional slow-freezing protocol because of uterine bleeding on the day of transfer. Three thawed embryos were transferred, and a normal male with an infant karyotype of 46,XY was delivered.

Conclusion: This case report demonstrates effective oocyte cryopreservation by vitrification.     

Effect of rescuing donated immature human oocytes derived after FSH/hCG stimulation following in vitro culture with or without Follicular Fluid Meiosis Activating Sterol (FF-MAS)—an embryo chromosomal and morphological analysis

Purpose: Studies in mice and humans have shown that Follicular Fluid – Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS. Methods: Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 μM or 20 μM. Seventy ±2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis. Results: Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups. Conclusion: Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results.     

Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification

Purpose: The purpose of this study was to determine the developmental ability and ultrastructure of MII mouse oocytes after cryopreservation by slow freezing or vitrification.Methods: Ovulated MII mouse oocytes were allocated to slow frozen, vitrified and control groups. Oocytes in the slow frozen and vitrified groups were cryopreserved using 1,2 propandiol (PROH) and ethylene glycol (EG) respectively as cryoprotectants. After thawing, the surviving MII oocytes in both cryopreserved groups and the control group were inseminated and their developmental ability was compared. The ultrastructure of MII oocytes in both cryopreserved groups was assessed immediately after thawing and 10 h post insemination at the pronuclear stage, and compared with that of the control group.Results: The survival rates were nearly identical in both cryopreserved groups. The fertilization rates were also identical and comparable to that of the control group. The further development of vitrified oocytes was similar to that of the control oocytes, whereas it was severely limited in the slow-frozen oocytes. In the slow-frozen MII oocytes, the intermediate filaments were destroyed and the oolemma and microvilli were also modified. At the pronuclear stage deterioration of mitochondria and the presence of numerous vacuoles were also observed within the ooplasm. In the vitrified MII oocytes, the intermediate filaments were the only structures affected and these cytoskeletal elements were reorganized at the pronuclear stage.Conclusions: Vitrification results in less ultrastructural damage and better post fertilization development of MII mouse oocytes than slow freezing.     

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage

Objective: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes.Design: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours.Setting: Infertility Medical Center at the CHA General Hospital, Seoul, Korea.Patient(s): Oocytes were obtained from patients undergoing gynecologic surgery.Main Outcome Measure(s): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin.Result(s): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3.Conclusion(s): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.     

多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

目的评价未成熟卵母细胞体外成熟（IVM）后形成的卵裂期胚胎经慢速冷冻一解冻后的发育能力。方法将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征（PCOS）合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组：复苏胚胎来源于体外成熟的卵母细胞组（IVM组,46例）和复苏胚胎来源于常规体内成熟的卵母细胞组（IVF组,339例）。采用慢冻速溶法解冻移植后比较两组患者的临床结局。结果IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66．67％;IVF组复苏胚胎1605枚,复苏后存活1082枚,复苏率为67．41％,两组比较,差异无统计学意义（P〉0．05）。IVM组患者的临床妊娠率和着床率分别为19．30％（11／57）和10．61％（14／132）,明显低于IvF组临床妊娠率（45．45％,175／385）和着床率（26．14％,240／918;P均〈O．05）。结论体外成熟卵母细胞发育形成的卵裂期胚胎慢速冷冻后临床结局欠佳,可能与冻融前胚胎自身的发育潜力有关。     

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring

Purpose: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. Methods: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activate with Sr ²⁺ -containing Ca ²⁺ -free medium. Results: After thawing, 75–85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. Conclusions: Mouse round spermatids can be cryopreserved for production of normal offspring.     

Improved maturation competence of ovarian tissue oocytes using a biphasic in vitro maturation system for patients with gynecological malignancy: a study on sibling oocytes

Purpose

To investigate the developmental competence of ovarian tissue oocytes from patients with gynecological tumors using a biphasic in vitro maturation system with capacitation (CAPA-IVM) in comparison with standard IVM.

Methods

This sibling pilot study included 210 oocytes in 10 patients with gynecological malignancies. After ovariectomies, ovaries were cut into even halves and immature cumulus-oocyte complexes (COCs) were retrieved from the ovarian tissue. COCs were separately cultured in either a biphasic CAPA-IVM system for 53 h or in standard IVM for 48 h. After IVM, all COCs were denuded and mature oocytes were either vitrified (N=5) or used for ICSI (N=5). Embryos were cultured for 5–6 days and obtained blastocysts were vitrified.

Results

Use of the CAPA-IVM system led to a higher meiotic maturation rate in ovarian tissue oocytes (OTO) compared to standard IVM (56 vs 35%, p=0.0045) and had a tendency to result in lower degeneration after IVM. Only the CAPA-IVM method supported blastocyst formation.

Conclusions

The biphasic in vitro maturation system improved the competence of OTO in comparison to the standard IVM method. The study suggests that fertility preservation programs could become more efficient using IVM after capacitation culture.

     

Follicle stimulating hormone effects on immature human oocytes: In vitro maturation and hormone production

Purpose: Our purpose was (1) to determine if in vitro maturation of unstimulated oocytes could be improved with the addition of urofollitropin; (2) to evaluate the output of estradiol, testosterone, progesterone, and androstenedione by the cultured oocyte-cumulus complex; and (3) to ascertain if steroid hormone production of the oocyte-cumulus complex correlates with final oocyte maturation stage. Methods: Fifty-eight immature oocytes were obtained from 11 regularly cycling women undergoing oophorectomy. The oocyte-cumulus complexes were randomly assigned to control medium (Ham’s F-10 supplemented with 7.5% fetal bovine serum) or test medium (control medium supplemented with 75 mIU/ml of urofollitropin). Results: (1) The addition of urofollitropin to oocyte culture medium does not significantly increase the ability of the oocyte to achieve the metaphase II stage; (2) the addition of urofollitropin significantly increases the production of progesterone, testosterone, and androstenedione by the oocyte-cumulus complex; and (3) there is no difference in the production of estradiol, progesterone, testosterone, and androstenedione by the oocyte-cumulus complex at the germinal vesicle, metaphase I or metaphase II stage of oocyte maturation. Conclusions: This information is of importance in the use of oophorectomy specimens for patients who must undergo an oophorectomy but desire to attempt pregnancy using their oocytes, in the use of oophorectomy specimens for donor oocytes, or for patients undergoing in vitro fertilization using immature oocyte collection.