Cryopreserved Immature Mouse Oocytes: A Chromosomal and Spindle Study

Purpose: Cryopreservation of human oocytes might provide an alternative approach to freezing supernumerary embryos obtained during IVF. This process, performed on immature denuded prophase I mouse oocytes, was investigated. Methods: We first investigated the capacity of frozen, immature, murine oocytes to continue in vitro maturation after thawing. We then evaluated the risk to offspring from chromosomal damage by cytogenetical and cytological (spindle) analysis. Finally, we attempted to determine the reasons for and the stage of maturation failure. Results: A total of 700 immature oocytes was frozen, 629 (90%) were recovered intact after thawing, and 53% extruded the first polar body, versus 74% for the control group. Freezing was not accompanied by an increase in aneuploidy in maturing oocytes (18 and 15% for thawed and control oocytes, respectively). Consequently, the first meiotic division occurred normally, without an increase in nondisjunction. Spindle analysis demonstrated only a few abnormalities (15 and 2% for thawed and control oocytes, respectively) incompatible with further development. Oocytes arrested during in vitro maturation were mainly at the metaphase I stage (64 and 76% for thawed and control oocytes, respectively). Whereas 17% of thawed oocytes were blocked before the formation of the first meiotic spindle, this never occurred in the control group. Conclusions: Immature murine oocytes can withstand cryopreservation, which is encouraging for future human application of this technique.     

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。     

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal

Purpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes. Methods: Oocytes that were at metaphase II about17–20 hr after insemination or intracytoplasmic sperm injection(ICSI) were treated with pronase to remove the zonapellucida and polar body (PB) and then spread on slides usingHCl and Tween 20. Two rounds of FISH were performedusing direct-labeled probes: chromosomes 1, 13, 21 (round1); chromosomes X, 7, 18 (round 2). Results: Of the 63 oocytes from 18 patients (mean age,32 years), 48 (76%) had one DNA complement as expected, 9(14%) had 2 DNA complements, 3 (5%) gave incomplete FISHsignals, and 3 (5%) were not analyzable. Of the 48 oocyteswith one set of DNA, 48% were haploid, 44% were aneuploidfor one or more chromosomes, and 8% were polyploid. Wealso found an increased frequency of predivision of chromatidbivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneousassessment of six chromosomes in human oocytes, and thereforecan be useful for accurately determining the incidence andcauses of genetic imbalances in human oocytes andapparently low fertilization rates.     

Human Embryos Derived from In Vitro and In Vivo Matured Oocytes: Analysis for Chromosomal Abnormalities and Nuclear Morphology

Purpose: To determine whether embryos resulting fromoocytes matured in vitro have a higher incidence of nuclearand/or genetic abnormalities compared to embryos resultingfrom oocytes matured in vivo. Methods: Fluorescence in situ hybridization analysis forchromosomes X, Y, and 18 was used to compare the ratesof aneuploidy, mosaicism, and nuclear abnormalities inembryos derived from oocytes that were prophase I ataspiration (immature group) to that observed in embryos resultingfrom oocytes that were metaphase I or II at aspiration(mature group). Results: Based on nuclear morphology, significantly moreembryos in the mature group (23percnt;) were classified as normalcompared to embryos in the immature group (3percnt;). Nodifference was found in the rate of aneuploidy or in the incidenceof mosaicism involving these chromosomes. Conclusions: These findings suggest that few embryosderived from prophase I oocytes collected following ovarianstimulation are morphologically normal.     

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.     

Fertility preservation for trans men: frozen-thawed in vitro matured oocytes collected at the time of ovarian tissue processing exhibit normal meiotic spindles

Purposes

At the moment of sex reassignment surgery (SRS), the ovarian tissue is sometimes cryopreserved as fertility preservation option for female-to-male trans men, also called trans men. During this preparation, cumulus-oocyte-complexes (COCs) can be found and in vitro matured. It is not known if these oocytes are developmentally competent. In order to use these oocytes for fertility preservation and subsequent fertilization, a normal spindle structure before and after vitrification is necessary.

Methods

A total of 680 COCs were collected from trans men (n = 16) at the time of SRS and after testosterone treatment. The COCs were subjected to in vitro maturation and those that reached the metaphase II stage (MII) were collected and split into two groups; group 1 was immediately fixed for spindle staining and group 2 was first vitrified and warmed followed by spindle staining. Statistical analysis was performed by Fisher’s exact test.

Results

After 48 h in vitro maturation, 38.1% of COCs were at MII stage. Those oocytes were split in two groups: (1) 126 MII oocytes in the noncryopreservation group and (2) 133 MII oocytes underwent cryopreservation through vitrification. The oocyte survival rate, after 2 h warming, was 67.7%. Both the noncryopreserved and the vitrified group showed comparable results concerning normal spindle structure and chromosomes alignment, 85.7% vs. 92.2% (P = 0.27).

Conclusions

Spindle structure analysis and chromosomal alignment after vitrification seem normal in in vitro matured COCs collected during the tissue processing of ovaries in trans men at the time of SRS. The MII oocytes do not seem to be morphologically affected by prolonged testosterone treatment.

     

Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes

OBJECTIVE: To analyze the chromosomal status of human embryos obtained from frozen-thawed oocytes. DESIGN: Fluorescence in situ hybridization analysis of embryos obtained after oocyte cryopreservation. SETTING: Department of Obstetrics and Gynecology at the University of Perugia, Italy, and the Instituto Valenciano de Infertilidad, Spain. PATIENT(S): Oocyte donors (n = 43). Fertilization, development, and chromosomal status of the embryos were compared with a control group (n = 18) of patients undergoing preimplantation genetic diagnosis for sex chromosome-linked diseases. INTERVENTION(S): Collection of oocytes after conventional ovarian stimulation and cryopreservation using propanediol as the cryoprotectant and a slow freezing procedure. Microinjection of surviving metaphase II oocytes and evaluation of fertilization and embryo development up to blastocyst stage. Chromosomal analysis after embryo biopsy. MAIN OUTCOME MEASURE(S): Survival, fertilization, and blastocyst rates. Embryo chromosomal analysis employing specific probes for chromosomes 13,18,21, X and Y. RESULT(S): The overall survival rate was 59.4%. There was no difference between cryopreservation and control groups in fertilization rates (76.5% vs. 90.5%) or blastocyst development (29.6% vs. 35%). The percentage of blastocysts from the original number of cryopreserved oocytes was only 5.6%, comparable to the 5.9% obtained in the control group. The percentage of embryos with abnormal number of chromosomes in the cryopreservation group (28.6%) was comparable to the 26% observed in the controls. CONCLUSION(S): Fertilization and cleavage rates after oocyte freezing are acceptable. Survival is, however, still poor, leading to overall results that make the technique clinically inefficient. There is no increase in the rate of chromosomal abnormalities, indicating that the technique is, nevertheless, safe enough to be further explored and improved.     

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.     

冻融体外受精-胚胎移植周期人未成熟卵母细胞的体外成熟及纺锤体结局

目的:探讨冻融的不同状态人未成熟卵母细胞体外成熟后纺锤体状态与受精率的关系。方法:随机收集本中心108个体外受精-胚胎移植周期(IVF)中183枚不同状态废气的成熟卵母细胞,分为:卵丘-卵母细胞复合物组,48枚;裸卵组,135枚,其中第一次减数分裂中期(MI)65枚,生发泡期(GV)70枚。玻璃化冷冻保存,经解冻、体外培养成熟后,应用Polscope成像系统观察纺锤体,然后行卵胞浆内单精子显微注射受精,记录各指标情况。结果:①卵丘-卵母细胞复合物组与裸卵组的存活率、体外成熟率、纺锤体出现率、受精率比较,均无统计学差异(P>0.05);②GV组的存活率显著高于MI组(P<0.05),而前者的体外成熟率显著低于后者(P<0.05);③各组体外成熟后有纺锤体出现的卵母细胞受精率均显著高于无纺锤体组(P<0.05,P<0.01)。结论:冻融IVF周期不同状态的人未成熟卵母细胞都有一定发育潜能;有纺锤体出现的冻融人未成熟卵母细胞质量较高。     

Cytogenetic Analysis of Sperm Nucleous Components of Iranian Normal and Sub-Fertile Individuals Using Zona Free Hamster Oocytes

Purpose: The purpose of this study was to investigate whether infertility is affected by sperm chromatin and cytogenetic abnormalities. To this purpose, the frequency of sperm premature chromosome condensation (PCC) induction and numerical chromosome abnormalities in the sperm of normal and sub-fertile men were analyzed. PCC rate was studied for evaluating the role of sperm chromatin abnormalities in the process of nuclear decondensation.Design: Controlled prospective study.Setting: Infertility Genetics Department, Royan Institute.Patient: Sub-fertile males who were referred for infertility treatment and sperm cytogenetical studies.Methods: Hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. Following treatment with Hyaloronidase, zona was removed by trypsin digestion. Sperms were classified according to the morphology, movement and counts and then processed by swim up method. After capacitation, zona-free oocytes were incubated with sperms, and then transferred to fresh media containing colcemid. Slides were prepared using Tarkowskies standard air-drying technique. Oocytes were analyzed using × 1000 microscope after staining in 5% of Giemsa.Main outcome measure: The incidence of sperm aneuploidy, PCC and penetration rate in three groups were determined.Results: Regarding the PCC rate, a significantly higher frequency was found in infertile patients. (P<0.001). The frequency of PCC in oligosperm samples was 36% compared to 19.37% in normal group. A higher frequency of numerical chromosome abnormalities was found in infertile patients. The rate of these abnormalities was 5.6% in normal group and 18.5% in oligospermic samples. Despite the considerable difference between those frequencies, this difference is not significant. (P>0.05)Conclusions: From the results it can be concluded that, formation of sperm PCC is a major cause of failed fertilization in individuals with sperm abnormalities. PCC may form due to chromatin abnormalities, improper DNA packing, chromosomal abnormalities and penetration delay of sperm. Also this may be involved in the etiology of some cases of idiopathic infertility. About numerical chromosome abnormalities although the differences are not significant, there is an association between sperm numerical chromosome abnormalities and male infertility. These abnormalities can be originated from meiotic process in spermatogenesis.     

Spindle organization after cryopreservation of mouse, human, and bovine oocytes

Oocyte cryopreservation would alleviate a number of ethical, social, and religious problems associated with human embryo storage. One potential problem is the effect of cryopreservation on the metaphase II spindle and chromosomes. The microtubules that make up the spindle tend to depolymerize at sub-physiological temperatures. Although there are numerous reports in the literature on this topic, discrepancies as to whether the spindle can or cannot reform persist. One of the confounding factors may be the low cryosurvival rates (around 50%) for mammalian oocytes. In recent years, a cryopreservation medium and protocol have been developed that allow oocytes of several species to be cryopreserved with high survival rates (>85%). Bovine, mouse, and human oocytes consistently reformed a morphologically normal spindle with chromosomes aligned along the metaphase plate (70% or higher) after first surviving cryopreservation (>87% survival for all species tested). Normal chromosome numbers were found in every second polar body tested by FISH (second polar bodies n = 4). It is concluded that the mammalian spindle, although depolymerized during cryopreservation, has the ability to reform, and in the mouse has been shown to function normally. Therefore, spindle reformation may not be a major cause for concern when storing mammalian MII oocytes.     

Polar body diagnosis of common aneuploidies by FISH

Purpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

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.     

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.     

Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope

OBJECTIVE: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. DESIGN: Morphological study. SETTING: Academic IVF clinic. PATIENT(S): Women undergoing oocyte retrieval for ICSI treatment. INTERVENTION(S): Oocytes were examined after in vitro maturation. MAIN OUTCOME MEASURE(S): The study examined meiotic spindle morphologic features and chromosome alignments. RESULT(S): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. CONCLUSION(S): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination.     

Chromosome investigation in in vitro fertilization failure

Purpose A chromosomal complement of 227 human oocytes was studied to provide information on the frequency and type of chromosomal abnormalities in oocytes failing in vitro fertilization.Results Normal haploid chromosome complement was found in 54.6%; chromosomal abnormalities consisting of diploid sets were identified in 16.7% and aneuploidy was observed in 26%. Premature condensation of sperm chromosomes of the G1-phase was observed in 22.9% oocytes. Male infertility was correlated with an increase in the rate of aneuploidy when compared with tubal infertility. The rate of chromosome abnormalities for the oocytes recovered from women who had no fertilized oocytes was significantly higher compared to those with at least one oocyte fertilized.Conclusion A high frequency of chromosome abnormalities in unfertilized oocytes suggests that natural selection against chromosome abnormalities may occur even prior to fertilization.     

有无卵丘细胞对玻璃化冷冻后小鼠卵母细胞细胞骨架及体外发育能力的初步研究

目的:探讨卵丘细胞在玻璃化冷冻中对小鼠卵母细胞发育潜能及细胞骨架的影响。方法:采用玻璃化冷冻技术,保存带卵丘细胞或完全剥除卵丘细胞的小鼠MⅡ期和GV期卵丘复合体/裸卵(COC/DO),复苏且GV卵体外培养成熟后分别作体外受精或免疫荧光标记检查纺锤体和染色体的完整性。结果:MⅡ-COC和GV-COC的复苏率均显著高于MⅡ-DO和GV-DO(分别为86.49%vs60.92%和85.94%vs64.93%,P<0.01)。GV-COC组的受精率、囊胚率均高于GV-DO组,且MⅡ-COC组和GV-COC组的纺锤体和染色体均正常率均分别高于MⅡ-DO组和GV-DO组,但无显著性差异(P>0.05)。结论:卵丘细胞在玻璃化冷冻卵母细胞中能有效减少冷冻对细胞骨架的损伤,并改善卵子复苏及胚胎发育潜能。     

The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes

OBJECTIVE: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI. DESIGN: The LC polscope was used to examine spindles in an observational study of living oocytes. SETTING: Academic IVF clinic. PATIENT(S): Women being treated for infertility. INTERVENTION(S): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. MAIN OUTCOME MEASURE(S): Spindle structure in living oocytes and fertilization after ICSI. RESULT(S): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. CONCLUSION(S): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI.     

Treatment variables in relation to oocyte maturation: Lessons from a clinical micromanipulation-assisted in vitro fertilization program

Objective: In an effort to understand the mechanism underlying the improved pregnancy rate observed in IVF cycles when gonadotropin-releasing hormone analogues (GnRH-a) are applied, we investigated a possible relationship between treatment variables and oocyte-nuclear maturity. Design: Nuclear maturity was retrospectively assessed in cumulus-free, denuded oocytes, obtained from women undergoing micromanipulation-assisted IVF treatment following controlled ovarian hyperstimulation with GnRH-a and menotropins. Setting: The setting was the infertility and IVF unit of a tertiary academic medical center. Participants: Two hundred twenty-one patients underwent 435 treatment cycles. Main Outcome Measure: This was the proportion of germinal vesicle-intact immature (GVII) oocytes. Results: One hundred fifty-four of the 3520 oocytes studied (4.4%) were in the GVII stage. These oocytes were found in 66 of the treatment cycles (15.2%) and in 54 of the patients (24.4%). Cycles in which GVII oocytes were detected did not differ from those in which all the aspirated oocytes were mature in the following respects: patient age, type and duration of infertility, controlled ovarian hyperstimulation protocol and time of ovum pickup. However, the GVII group was characterized by a significantly higher peak estradiol level, as well as a higher number of mature follicles visualized sonographically (diameter, >14 mm) and oocytes retrieved. Conclusions: Comparing the present findings with previously published data, it appears that the inclusion of GnRH-a in the stimulation regimen is associated with a lower proportion of immature oocytes. A higher occurrence of oocyte-nuclear immaturity is apparently associated with a significantly better ovarian response to stimulation. The high incidence of immature oocytes observed in patients with normospermic partners and low fertilization rates in previous cycles may suggest that the fertilization failure in some of these cases is due to oocyte, rather than sperm, dysfunction.