人黄体期卵母细胞的体外成熟及玻璃化冷冻的实验研究

目的:探讨未成熟卵母细胞体外成熟(IVM)技术联合玻璃化冷冻保存黄体期卵母细胞对某些女性肿瘤患者的生育能力的保存情况。方法:采集因妇科肿瘤等行卵巢切除手术过程中穿刺获取的256枚未成熟卵母细胞,按取卵时患者的月经周期分为卵泡期组(143枚)与黄体期组(113枚),每组再随即分为新鲜对照组及玻璃化冷冻组。分别进行IVM后行新鲜卵胞质内单精子注射(ICSI)授精和玻璃化冻融卵ICSI授精,比较各组间IVM后MII卵率、受精率、卵裂率、优质胚胎率。结果:①卵泡期与黄体期卵母细胞的IVM率差异无统计学意义;而组间复苏存活率(68.0%vs 48.1%)有统计学差异(P<0.05);卵泡期与黄体期卵母细胞新鲜组间受精率、卵裂率、优质胚胎率无统计学差异,冷冻组间差异亦无统计学意义。②与新鲜组相比,玻璃化冷冻使卵泡期与黄体期卵母细胞的受精率均降低(P<0.01)。结论:黄体期未成熟卵母细胞可以体外成熟并有继续发育为优质胚胎的能力;玻璃化冷冻使卵母细胞受精率、卵裂率下降。IVM和冻融后体外授精是某些女性肿瘤患者保存生育能力的一种有临床应用前景的方式。     

Pregnancy following transfer of ooplasm from cryopreserved-thawed donor oocytes into recipient oocytes

OBJECTIVE: To determine if frozen-thawed donor oocytes could be used to provide cytoplasm for transfer into patients' oocytes to improve subsequent embryonic development. DESIGN: Prospective evaluation of the procedure in consenting IVF patients. SETTING: Assisted reproductive technology program. PATIENT(S): The study was open to consenting IVF patients (of any age) with a history of poor embryo quality or those couples in which the wife's age was > or = 40 years. INTERVENTION(S): Transfer of donor egg cytoplasm from frozen-thawed oocytes into the oocytes of infertile recipients. MAIN OUTCOME MEASURE(S): Donor oocyte survival following cryopreservation, fertilization following cytoplasmic transfer into recipient oocytes, embryo quality, and pregnancy outcome. RESULT(S): Oocytes collected from four donors were cryopreserved and 61% (28/46) survived the thaw procedure. Cytoplasmic transfer was performed on the eggs of four patients, with fertilization occurring in 70.3% (26/37). Twin pregnancy was established in one patient (35 years of age) with a history of poor embryo quality. CONCLUSION(S): Cryopreserved donor oocytes may provide a source of cytoplasm for transfer into recipient oocytes, eliminating the need for cycle synchronization between donor and infertile patient.     

Oocyte-induced haploidization

This paper describes the technical approach to treatment of age-related oocyte aneuploidy. Although one solution can be oocyte/embryo selection, another is represented by the nuclear transplantation procedure. The efficiency of nuclear transplantation into immature oocytes is described as a way of generating embryos, and the possibility that viable female gametes can be constructed by transfer of diploid somatic cell nuclei into enucleated oocytes. Germinal vesicle (GV)-stage mouse oocytes were collected from unstimulated ovaries and somatic nuclei were obtained from mouse cumulus cells obtained after ovarian stimulation. Spare human GV-stage oocytes were donated from consenting patients undergoing intracytoplasmic sperm injection (ICSI) treatment, and human somatic cells were stromal cells coming from uterine biopsies performed on consenting patients undergoing endometrial cell co-culture. GV ooplasts, prepared by enucleation, were transplanted with either GV or somatic nuclei by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were selected at random for insemination or cytogenetic analysis. GV transplantation was accomplished with an overall efficiency of approximately 80 and 70% in the mouse and the human respectively. The maturation rate of 96% (mouse) and 62% (human) following reconstitution was comparable to that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%. After the transfer of mouse cumulus or human endometrial cell nuclei into enucleated immature oocytes, a polar body was extruded in >40%. In a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, the chromosomes segregated normally at the time of polar body extrusion. The technique of nuclear transplantation itself did not increase the incidence of chromosomal anomalies in the mouse or human, since their oocytes reconstituted with homologous donor GV resumed meiosis to metaphase II and maintained a normal ploidy. In addition, immature mouse ooplasts induced haploidization of transplanted somatic cell nuclei. Although further evaluation of their genetic status is needed, the procedure may offer a realistic way of producing normal oocytes in cases of aged-related infertility. While the procedure is technically similar to cloning, it would generate a unique individual as a result of the contribution of both parental genomes.     

Correlation of somatic cell steroid secretion and quality of generated oocytes after in-vitro stimulation of mouse follicles

Purpose: To test the possibility of follicular somatic cell steroidogenesis as a marker for quality of their embraced oocytes. Methods: Mechanically isolated mouse preantral follicles were cultured and matured in-vitro (IVC/IVM) for study. Results: During IVC/IVM, oogenesis occurred concomitantly with folliculogenesis in a coordinated manner and simultaneously with progressive increments of somatic cell steroidogenesis. Follicular E₂ production of matured oocytes were significantly higher than that of immature ones. The majority of MII oocytes (32/36) and all developed blastocysts(12/12) were associated with active E₂ production prior to ovulation. In this study, 18 MII oocytes met both requirements for active and optimal E₂ production. 13 of them were fertilized and 10 developed into blastocysts. Conclusion: Active somatic cell steroidogenesis prior to ovulation and an optimal steroid milieu at ovulation are prerequisites for generation of competent oocytes after follicular maturation in-vitro.Predictability of somatic cell steroidogenesis for oocyte competency after follicular IVC/IVM.     

体外成熟卵母细胞体外受精方式的探讨

目的:探讨经体外成熟卵母细胞是否可采用常规IVF方式授精。方法:自愿捐献的常规废弃的未成熟卵母细胞经体外成熟后的卵母细胞206个,随机分为3组:A组(n=69),授精时尽可能保存卵丘细胞并行常规IVF授精;B组(n=68),授精前将卵丘细胞完全拆除后行常规IVF授精;C组(n=69),将卵丘细胞完全拆除后行ICSI授精。常规IVF授精的精/卵个数比约为50 000∶1。比较3组卵子的受精率、卵裂率和优质胚胎率。结果:A组与C组在受精率(82.61%vs 85.51%)、卵裂率(94.74%vs 94.92%)、优质胚胎率(42.59%vs 46.43%)3个方面均无统计学差异(P>0.05),A组、C组的受精率、卵裂率均显著高于B组(14.71%,50.00%)(P<0.01),B组没有优质胚胎出现。结论:体外成熟后的卵母细胞在卵丘细胞较完整时可进行常规IVF方式授精。     

Effects of in-vitro or in-vivo matured ooplasm and spindle-chromosome complex on the development of spindle-transferred oocytes

To study the effects of in-vitro matured ooplasm and spindle-chromosome complex (SCC) on the development of spindle-transferred oocytes, reciprocal spindle transfer was conducted between in-vivo and in-vitro matured oocytes. The reconstructed oocytes were divided into four groups according to their different ooplasm sources and SCC, artificially activated and cultured to the blastocyst stage. Oocyte survival, activation and embryo development after spindle transfer manipulation were compared between groups. Survival, activation, and cleavage rates of reconstructed oocytes after spindle transfer manipulation did not differ significantly among the four groups. The eight-cell stage embryo formation rates on day 3 and the blastocyst formation rate on day 6 were not significantly different between the in-vitro and in-vivo matured SCC groups when they were transplanted into in-vivo matured ooplasm. The rate of eight-cell stage embryo formation with in-vitro matured ooplasm was significantly lower (P < 0.05) than that of embryos with in-vivo matured ooplasm, and none of the embryos developed to the blastocyst stage. Therefore, SCC matured in vitro effectively supported the in-vitro development of reconstructed oocytes. Ooplasm matured in vitro, however, could not support the development of reconstructed oocytes, and may not be an appropriate source of ooplasm donation for spindle transfer.     

Decreased in vitro fertilization and cleavage rates after an equipment error during CO(2) calibration

Objective: To report the decreased IVF rate of oocytes and the reduced cleavage rate of pronuclear stage embryos after gas has backflowed from a gas analyzer into an incubator during the calibration of the CO₂ concentration.

Design: Case report.

Setting: Clinical research unit for reproductive medicine in a hospital.

Intervention(s): Backflow of gas from a gas analyzer into an incubator during calibration of the CO₂ concentration.

Main Outcome Measure: The IVF rate of oocytes and the cleavage rate of pronuclear stage embryos.

Result(s): Thirty-two oocytes were retrieved from four infertile female patients. Nine oocytes were fertilized and four fertilized oocytes were cleaved. In a related animal experiment, the cleavage rate was 57.1% (36/63) in the control group and 25.4% (16/63) in the study group (P = .00026).

Conclusion(s): The backflow of gas from the gas analyzer adversely affected the fertilization of oocytes and the cleavage of pronuclear stage embryos.     

Effects of cyclophosphamide administration on the in vitro fertilization of mice

Purpose

To evaluate the oocyte fertilization ability and embryo growth after cyclophosphamide (CPA) treatment in mice.

Methods

Mice were treated with CPA at different doses (0‐800 mg/kg body weight). The oocytes then were retrieved and evaluated for their in vitro fertilization efficiency.

Results

The average number of metaphase II (MII) oocytes significantly decreased by ≥400 mg/kg CPA administration. The fertilization rate also decreased in the group that was treated with ≥400 mg/kg CPA. However, after fertilization, the embryos demonstrated normal growth ability. Two weeks after CPA administration, the number of mice from which the oocytes could be retrieved markedly decreased, but the fertilization rate and development of morphological features in the embryos were similar to those of the controls. One month after CPA administration, the number of mice from which the oocytes could be retrieved, fertilization rate, and development of the morphological features in the embryos were similar to those of the controls.

Conclusion

The number of oocytes decreased as the CPA administration level increased; however, the oocytes' potential for fertilization and development to the blastocyst stage was not significantly affected. One month after CPA administration, the number of oocytes and the potential for development into blastocysts were recovered.     

Demonstration of chlorinated hydrocarbons in follicular secretions]

To determine the concentration of HCB, HCH and PCB in human follicular fluid, 315 preovulatory follicular taken from 45 patients and aspirated by transvaginal follicular puncture were analysed with the aid of gas chromatography. We distinguished follicular fluid without oocytes and follicular fluid with oocytes. Again the latter is subdivided into follicular fluid containing secondarily fertilized oocytes and non-fertilized oocytes. There are great differences in HCB (0.08 - 1.87 ng/ml), HCH (0.05 - 1.0 ng/ml) and PCB (0.28 - 2.11 ng/ml) concentrations, varying even in the follicles of the same patient. Contrary to all our expectations we did not find any significant difference in the HCB, HCH and PCB concentration of the different follicular fluids. A direct influence of these substances on the fertilization of oocytes in IVF-ET-procedure could not be established.     

Metaphase II karyoplast transfer from human in-vitro matured oocytes to enuclueated mature oocytes

Metaphase II karyoplast transfer is believed to be a useful method to rescue aged oocytes. This study attempted karyoplast transfer of in-vitro matured metaphase II (MII) oocytes, as a model of aged oocytes, into enucleated freshly ovulated metaphase II oocytes with visualization of their chromosomes under an inverted microscope. Recipient karyoplasts derived from immature oocytes were cultured in-vitro until first polar body extrusion. After 1–2 days culture, 52.1% extruded a polar body, 95.5% had PSC, aneuploidy was very low (4.5%) and none had structural aberrations. Donor oocytes were obtained from IVF or intracytoplasmic sperm injection (ICSI) patients. Chromosomes were easily confirmed in 92.3% and 95.0% of in-vivo and in-vitro matured oocytes respectively. Thirty-one karyoplasts were placed in the perivitelline space of enucleated donor oocytes, and 25 (80.6%) fused to form a reconstituted oocyte. Fertilization, cleavage and blastocyst formation rates following ICSI were 76.0%, 64.0% and 28.0% respectively for reconstructed oocytes and 59.2%, 48.0% and 3.1% respectively for control (in-vitro matured) oocytes. Chromosomal analysis of five embryos developed after karyoplast transfer and ICSI showed normal diploid sets of 46 chromosomes. In conclusion, this metaphase II karyoplast transfer technique can be applied to the solution of chromosomal abnormalities related to oocyte ageing.     

Application of intra- and extracellular sugars and dimethylsulfoxide to human oocyte cryopreservation

Purpose  Oocyte cryopreservation may avoid many complications of human embryo freezing and provide future fertility for women undergoing cancer therapy. The objective of this study was to explore the application of intra- and extracellular sugars in combination with small amounts of dimethylsulfoxide (DMSO) to human oocyte cryopreservation as an alternative approach. Methods  Discarded human oocytes that were obtained from IVF patients under informed consent and IRB approval, were cryopreserved by slow cooling to −196°C after being randomly distributed into three groups: (i) DMSO control without intra- and extracellular sugar; (ii) extracellular sugar (raffinose) + DMSO; and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) + DMSO. Subsequently, all cryopreserved oocytes were thawed rapidly, and their survival was assessed by morphological criteria after 24 h of culture. Results  A total of 71 oocytes were evaluated in three groups with survival rates of 88.5% (23/26), 68.2% (15/22), and 52.2% (12/23) for intra- and extracellular sugar+DMSO, extracellular sugar+DMSO, and DMSO control groups, respectively. Conclusion  These results support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes.     

Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures.

OBJECTIVE: To assess the technique of injecting a single sperm and cytoplasm obtained from tripronucleate zygotes into metaphase II oocytes for the treatment of patients with repeated implantation failure after intracytoplasmic sperm injection or IVF. DESIGN: Clinical study. SETTING: Private infertility clinic. PATIENT(S): Patients with repeated implantation failure after intracytoplasmic sperm injection or IVF. INTERVENTION(S): The metaphase II oocytes of recipients were injected with their husbands' spermatozoa and cytoplasm aspirated from the tripronucleate zygotes of donors. MAIN OUTCOME MEASURE(S): Fertilization after cytoplasm and sperm injection, embryo development, and successful pregnancy. RESULT(S): In total, 62 metaphase II oocytes from nine recipients were injected. Of the 62 injected oocytes, 3 (5%) degenerated and 43 (69%) had two pronuclei 18 hours after injection. Thirty-nine oocytes with two pronuclei cleaved to the two-cell to six-cell stage after another 24 hours of culture. All cleaved embryos were transferred into the uteruses of recipients. Four clinical pregnancies occurred in four recipients. No abnormal chromosomes were observed after amniocentesis and karyotyping in all pregnancies. Five healthy infants were born. CONCLUSION(S): Injection of the cytoplasm of tripronucleate zygotes may enhance the clinical pregnancy rate in patients with repeated implantation failure after intracytoplasmic sperm injection or IVF.     

多囊卵巢综合征患者卵丘形态与未成熟卵母细胞发育潜能关系

目的 研究多囊卵巢综合征患者无刺激周期取出的不同形态未成熟卵母细胞的发育潜能。方法 43例PCOS不孕患者进行了47个未成熟卵母细胞体外成熟培养（IVM）周期。所有患者均未经促卵泡素刺激,予以HCG36h后取卵。根据取出的卵-冠-丘复合物形态将其分为3组：卵丘紧密组、卵丘松散组、无卵丘组。比较3组的体外成熟率、受精率和优质胚胎率。结果 47个IVM周期共收集未成熟卵母细胞874枚,体外成熟率61.19%,受精率71.07%,着床率13.13%。卵丘松散组的体外成熟率明显高于卵丘紧密组（72.26%vs49.54%,P〈0.05）,受精率、优质胚胎率三组间无差异。结论 PCOS患者无刺激周期取出的未成熟卵母细胞中,卵丘松散、扩张的卵母细胞具有更好的体外成熟潜力。     

The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei

Objective: To compare the oocyte-activating ability of whole human round spermatids and their isolated nuclei.

Design: Prospective study using sibling oocytes from patients undergoing spermatid conception and intracytoplasmic sperm injection treatment cycles.

Setting: Private assisted reproduction laboratories and a university department.

Patient(s): Couples with male infertility.

Intervention(s): Sibling oocytes were injected either with whole round spermatids or with their isolated nuclei, followed by artificial triggering of oocyte activation with calcium ionophore. Other sibling oocytes were injected either with isolated spermatid cytoplasm or with whole mature spermatozoa.

Main Outcome Measure(s): Numbers of activated oocytes and cleaving embryos.

Result(s): After oocyte activation was boosted with calcium ionophore, whole spermatids and isolated spermatid nuclei were equally effective in supporting oocyte activation and the formation of pronuclei, whereas no control oocyte was activated under the same conditions after injection of isolated spermatid cytoplasmic compartments. Cleavage rates were lower after the injection of isolated spermatid nuclei than after the injection of whole spermatids.

Conclusion(s): The factor responsible for human oocyte activation after round spermatid injection is associated with spermatid nuclei. The requirement for the artificial trigger (calcium ionophore) suggests that this factor is identical to the male gamete activity previously characterized as calcium oscillator.     

Fertilization of bovine oocytes induced solely with combined laser microbeam and optical tweezers

Purpose: Our purpose was to show that fertilization of oocytes can be obtained solely by laser light-mediated manipulation of gametes. Method: A small channel was drilled into the zona pellucida of bovine oocytes using an ultraviolet (UV)-laser microbeam. Highly diluted cattle sperm were not able to fertilize the laser drilled oocytes. Results: Fertilization was achieved only when three to five cattle sperm were trapped with optical tweezers and inserted directly through the laser drilled hole into the perivitelline space. After 20 hr, 3 of 79 (3.8%) oocytes revealed two pronuclei and a sperm tail within their cytoplasm. Cattle sperm are difficult to catch. Therefore, the gametes had to remain for about 20 min in room atmosphere, which might be the reason for the low fertilization results. Conclusions: The results indicate that a combined UV-laser microbeam and optical tweezers trap can be used successfully for noncontact microinsemination procedures.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, April 3–7, 1995, Vienna, Austria.     

Improvement of embryonic development and production of offspring by transferring meiosis-II chromosomes of senescent mouse oocytes into cytoplasts of young mouse oocytes

Purpose  The effects of reciprocal transplantation of meiosis-II chromosomes between senescent and young mouse oocytes were evaluated based on pre- and post-implantation development ability of resultant embryos. Methods  Karyoplasts including meiosis-II chromosomes of oocytes from senescent Rockefeller mouse/Ms-Rb(6, 15) females (10 to 12 months, age-related infertile mice) were transferred into cytoplasts of oocytes from young F₁ females (3 to 5 months). Reconstructed oocytes were fertilized in vitro, and then the resultant embryos were cultured in vitro and transferred to recipient mice. Results  The reconstructed oocytes that consisted of aged-karyoplasts and young-cytoplasts showed significantly improved embryonic development (from 23.2% to 30.0%) and development to term (from 6.3% to 27.1%, P < 0.05) as compared with the oocytes reconstructed from young-karyoplasts and aged-cytoplasts. Conclusions  The present study showed successful rejuvenation for age-related infertility using transplantation of meiosis-II chromosomes in animal experimental models. Capsule Transplantation of meiosis-II chromosomes of senescent mouse oocytes into cytoplasts of young mouse oocytes improves subsequent embryonic and fetal development until birth.     

Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration

Partial zona dissection (PZD), a method using mechanical force to open the human zona pellucida, and zona drilling, which uses acidic Tyrode's (AT) medium, were compared in 1-day-old oocytes prior to reinsemination. The incidences of monospermy and polyspermy were 13/54 (24%) and 14/54 (26%) following PZD and 6/46 (13%) and 8/46 (17%) following the use of AT medium. This compared favorably with conventional reinsemination: 15/161 (9%) monospermy and 4/161 (3%) polyspermy. Three of the 27 PZD embryos became blastocysts, while none of the AT-exposed embryos developed satisfactorily. Eleven male-factor couples had some of their oocytes randomly treated with PZD prior to insemination; each of the patients had non-micromanipulated control oocytes. Monospermic fertilization and cleavage (23/34; 68%) doubled (P less than 0.05) when PZD was compared with the control oocytes (10/30; 33%). Replacing two PZD and a single control embryo in two patients resulted in twin pregnancies. A third twin pregnancy was established following replacement of only micromanipulated embryos.     

Maturation and apoptosis of human oocytes in vitro are age-related

Objective: To study the morphological changes of apoptotic oocytes, and rates of in vitro maturation and apoptosis of human oocytes in relation to age.

Design: Prospective comparative study.

Setting: Reproductive medicine center.

Patient(s): Women undergoing surgery for ovarian cysts.

Intervention(s): Oocytes were incubated in Ham’s F-10 medium with 15% fetal cord serum (FCS) for 32 to 120 hours and were examined under inverted microscope every 6 to 8 hours.

Main Outcome Measure(s): Oocyte maturation and apoptosis, Fas antigen.

Result(s): The morphologic changes characteristic of apoptosis oocytes were shrinkage, or the occurrence of cytoplasmic condensation, membrane blebbing, fragmentation of the oocyte into “apoptotic” bodies of unequal size, or internucleosomal DNA cleavage as shown by TUNEL. The maturation rates of oocytes were highest in those from women aged 21 to 30 years, and lowest in those aged 41 to 50 years. Apoptosis occurred in 17.1% (age group 21 to 30 years), 37.7% (31 to 40 years), and 52.3% (41 to 50 years). The rate of apoptosis of human immature oocytes cultured in vitro was significantly higher in those from older women who were 41 to 50 years old than in those women 21 to 40 years old. Fas antigen was found to be present on apoptotic oocyte membranes.

Conclusion(s): The developmental potential of oocytes from older women decreased in vitro in a manner similar to that seen in vivo. DNA fragmentation in oocytes associated with apoptotic death might be one of the reasons for poor oocyte quality and lower fertility in older women. Fas antigen in the oocyte presumably mediates apoptosis.