In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.     

Lack of effect of high-dose antioestrogen on the maturation and in-vitro fertilization of human oocytes

Thirty-four women requesting laparoscopic sterilization underwenta fixed schedule regimen for multiple follicular developmentwhich included norethisterone and clomiphene citrate. Follicleaspiration for oocyte recovery was attempted laparoscopically34 h after administration of 5000 IU human chononic gonadotrophin(HCG). Nineteen women were given 80 mg tamoxifen orally 4 hprior to HCG injection, while 15 acted as controls. There wasno statistical difference in fertilization rates in vitro betweentamoxifen-treated patients and controls (80 and 68% respectively).In addition, the morphological characteristics of the oocytes,the rates of cleavage, and the concentrations of oestradiol,progesterone and androstenedione in follicular fluid were similarin the two groups. Tamoxifen was detected in substantial amountsin follicular fluids of patients given tamoxifen. These resultssuggest that high-dose tamoxifeii, in clinically used doses,does not adversely affect the final stages of maturation orthe fertilization and early cleavage of human oocytes.     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching

Immature oocyte recovery followed by in-vitro oocyte maturationand in-vitro fertilization is a promising new technology forthe treatment of human infertility. The technology is attractiveto potential oocyte donors and infertile couples because ofits reduced treatment intervention. Immature oocytes were recoveredby ultrasoundguided transvaginal follicular aspiration. Oocyteswere matured in vitro for 36–48 h followed by intracytoplasmicsperm injection (ICSI). Embryos were cultured in vitro for 3or 5 days before replacement. Assisted hatching was performedon a day 5 blastocyst stage embryo. Embryo and uterine synchronywere potentially enhanced by luteinization of the dominant follicleat the time of immature oocyte recovery. Mature oocyte and embryoproduction from immature oocyte recovery were similar to theprevious IVF results of the patients. A blastocyst stage embryo,produced as a result of in-vitro maturation, ICSI, in-vitroculture and assisted hatching, resulted in the birth of a healthybaby girl at 39 weeks of gestation.     

Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection

This case report describes the birth of a baby following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) carried out on the second day after oocyte pick-up of in-vitro-matured metaphase I and germinal vesicle stage oocytes. The couple had a history of three failed intrauterine insemination attempts and reduced fertilization rates in two previous in-vitro fertilization (IVF) cycles. In the IVF-ICSI treatment cycle, 6/11 mature oocytes became fertilized following ICSI on the first day. However, the patient failed to conceive following the transfer of three embryos. Five oocytes were immature (two at metaphase I stage and three with a germinal vesicle) and these were cultured overnight. All had extruded a polar body by the following day and ICSI was therefore performed; four oocytes became fertilized, and were cryopreserved at the pronulear stage in propanediol. In the next treatment cycle, transfer of frozen embryos was planned. The pronuclear zygotes were thawed and cultured for 24 h prior to the transfer of two embryos in a cycle stimulated with low doses of follicle stimulating hormone. This resulted in a pregnancy and the delivery of a healthy baby boy. In-vitro maturation of metaphase I and germinal vesicle oocytes which are routinely collected in IVF-ICSI cycles, followed by second day ICSI fertilization, may provide a valuable source of embryos for infertile couples.      

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.     

Timing of in-vitro fertilization of cumulus-free and cumulus-enclosed human oocytes

In humans, in contrast to other species, sperm capacitationrequires a very short time, as in-vitro fertilization has beenobtained after only 45 mm of contact between oocytes and spermatozoacapacitated for 1 h. No fertilization occurred, whatever theduration of sperm capacit.ation, when gamete mixing did notexceed 30 mim. On the contrary, 85% of cumulus-free mature oocytesexposed to sperm for 1–4 h were fertilized. The presenceof the pre-ovulatory, fully-expanded or compact cumulus massdid not represent a physical barrier to sperm progression, aswe observed no delay in fertilization when oocytes were enclosedin the cumulus. The use of a short insemination protocol (1–4h instead of 17–20 h) did not reduce the fertilizationrate of denuded or cumulus-enclosed oocytes and had no significanteffect on the morphological appearance of the embryos or theircleavage rates.     

Infertility: Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients

A study was conducted on patients who had attempted and failedprevious in-vitro fertilization (IVF) procedures an averageof 3.8 times following the application of assisted hatchingwith conventional culture systems. The aim of this investigationwas to determine if addition of co-culture methodologies couldreduce embryonic abnormalities and thus improve the prognosisfor pregnancy. The study population consisted of 123 patients,subdivided into three patient categories. Previous IVF resultsfrom conventional culture were used to evaluate any potentialbenefits derived from the present co-culture application. Followingco-culture, the rate of blastomere development was increasedand the rate of fragmentation decreased. An increased rate ofblastomere development was most noticeable in the patients aged39 years with no male factor as well as the intracytoplasmicsperm injection (ICSI) subgroup. Similarly, the rate of fragmentationwas significantly reduced in the aforementioned subgroups. Themost pronounced impact of co-culture was on pregnancy and implantationrates. The overall clinical and ongoing pregnancy rates were38% (47/123) and 36% (44/123) respectively. The correspondingimplantation rate was 17% (72/412) as shown by embryonic cardiacactivity. The ongoing pregnancy rates in the 39 years no malefactor, 40 years no male factor and ICSI no age limit patientsubgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively.The results indicate that addition of co-culture to the IVFprocedure for poor-prognosis patients may be advisable.     

Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization

A total of 101 multipronuclear oocytes (7.4% of fertilizations)were retrospectively identified in this in-vitro fertilizationprogramme. The use of a manual syringe suction system, insteadof an electric pump, to aspirate follicles, was associated witha significant increase in the proportion of oocytes with fracturedzonae pellucidae (P < 0.001), a lower normal fertilizationrate (P < 0.05) and a higher proportion of multipronuclearfertilizations (P < 0.001). Irrespective of the mode of follicularaspiration, significantly more multipronuclear fertilizationsoccurred following stimulation with a combination of clomipheneand human menopausal gonado-trophin, than after clomiphene alone(P < 0.05). It was concluded that the aspiration pressures,created by syringe suction, were more likely to rupture thezona pellucida of some oocytes, while in others it predisposedto an increased multipronuclear fertilization rate.     

Fertilization and early embryology: Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization

A preliminary study and related clinical trial were performedto evaluate the effects of granulosa-lutein cell co-cultureon human embryo development and pregnancy rates for in-vitrofertilization (IVF). In the study, sibling two-pronuclear zygoteswere randomly allocated to culture with (co-culture) or without(control) autologous granulosalutein cells. After 24 h, embryoswere examined for blastomere number and degree of fragmentation.Co-culture had no effect on the average number of blastomeresper embryo at 24 h; however, fragmentation was significantlydecreased in co-cultured embryos (0.7 ± 0.1) comparedwith controls (1.3 ± 0.2; P < 0.05). In the subsequentclinical trial, all two-pronuclear zygotes were co-culturedfor 48 h prior to embryo transfer. The live birth rate per embryotransfer was 43.4% with an implantation rate per embryo of 17.6%.Of the untransferred embryos, 68% developed to the blastocyststage and were cryopreserved. We conclude that the simple systemof autologous granulosa-lutein cell co-culture improves embryodevelopment, implantation and subsequent pregnancy rates forIVF.     

Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes

BACKGROUND: With improved prospects for the use of human oocyte in-vitro maturation in assisted reproductive technologies, the need to define more clearly the coordination of nuclear and cytoplasmic maturation has arisen. METHODS: Immunofluorescence and confocal microscopy were used to evaluate cell cycle-dependent modifications in chromatin and microtubules in human germinal vesicle oocytes (n = 455) undergoing in-vitro maturation. RESULTS: Four distinct classes of germinal vesicle stage oocytes were identified based on the expression of G2/interphase characteristics, but, of these, only one class of oocytes was competent to complete meiotic progression to metaphase-II in vitro. The majority of germinal vesicle stage oocytes resumed meiosis within 6 h (88.9%) of culture and exhibited an accelerated pace of progression to metaphase-II (66.7%) over 24 h, but in general were unable to maintain meiotic arrest and defaulted into interphase within 24 h of polar body emission. Characterization of microtubule dynamics and chromatin phosphorylation demonstrates specific cell cycle deficiencies in in-vitro matured human oocytes. CONCLUSION: This work forms a basis for future studies aimed at optimizing nuclear and cytoplasmic maturation during in-vitro maturation.     

Ovary and ovulation: In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

A simplified culture system was developed for the in-vitro maturationof early preantral mouse ovarian follicles. The follicles werecultured singly in 20 (µ droplets under oil in mediumsupplemented with recombinant follicle stimulating hormone (r-FSH)at 37°C and 5% CO² in air. The follicles grew and becameattached to the bottom of the dish, progressively lost theirspherical structure by outgrowth of the granulosa cells throughthe basal membrane and developed follicles with antral-likecavities. The normal three-dimensional follicular structurewas lost but all components, i.e. theca, granulosa and oocyte,remained functional, as was proven by the oestradiol, inhibinand progesterone secretion patterns. Follicle survival exceeded80% and histological analysis proved the absence of atresiaand cell death in granulosa cells up to day 16. Oocytes of 55(± 4) µm diameter on the day of isolation reached74 (± 3) µm by day 16 of culture. The optimal momentfor inducing the final meiotic maturation with human chorionicgonadotrophin was investigated: the highest absolute numbersof metaphase II oocytes were obtained on days 12 and 14 (39and 41%). The fertilizing potential of the in-vitro maturedoocytes was comparable to in-vivo matured controls. A 50% hatched-blastocystdevelopment rate was observed.     

Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization

At 16–18 h after insemination, 5.5% of the inseminatedoocytes displayed only one pronucleus. The incidence of single-pronucleatedoocytes was not correlated with the age of the patient. Cytogeneticanalysis of 41 embryos derived from single-pronucleated oocytesrevealed a haploid chromosome complement in 12.2%, a diploidchromosome complement in 80.5% and a triploid set in 7.3% ofthe embryos. In one diploid metaphase, a Y chromosome couldbe clearly demonstrated. A total of 312 single-pronucleatedoocytes were evaluated twice (at 16–18 h and at 20–24h after insemination). In 25% of the single-pronucleated oocytesa second pronucleus was observed 4–6 h later, suggestingasynchronous or delayed pronuclear formation. The cleavage ofthese embryos was similar to the cleavage of embryos with twopronuclei at the first evaluation. Parthenogenetic activationand asynchronous pronuclei may both be mechanisms leading tothe morphological observation of a single pronucleus. In clinicalpractice, the second repeat observation of single-pronucleatedoocytes became part of the standard procedure.     

Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection

Human oocyte cryopreservation results in poor survival and subsequentfertilization rates. It has been suggested that freeze-thaw-inducedchanges in the zona pellucida may impair sperm penetration orattachment. The aim of this study was to compare fertilizationand cleavage rates in cryopreserved oocytes inseminated by conventionalin-vitro fertilization (IVF) or intracytoplasmic sperm injection(ICSI). A total of 220 oocytes, obtained from volunteers whohad undergone ovarian stimulation, were cryopreserved usinga slow freeze-rapid thaw protocol with 1.5 M propanediol asthe cryoprotectant. Surviving oocytes (n= 74, 34.4%) were randomlyallocated for fertilization by conventional IVF (group 1) orICSI (group 2) using cryopreserved spermatozoa from a singledonor of proven fertility. Fertilization was achieved in five(13.5%) of the oocytes in group 1 and 17 (45.9%) in group 2(P < 0.005), with only one oocyte in group 1 exhibiting normalfertilization as opposed to 16 (43.2%) in group 2 (P < 0.001).Similarly, one oocyte fertilized by IVF cleaved, while all fertilizedwith ICSI cleaved (P < 0.001). We conclude that althoughthe survival of oocytes is poor following cryopreservation,fertilization and cleavage rates can be enhanced significantlyusing ICSI. These data also suggest that the method of cryopreservationused in this study affected the zona pellucida, such that normalsperm attachment or penetration was impaired.     

A simple method for in vitro maturation,in vitro fertilization,and co-culture of bovine oocytes

Summary An efficient procedure has been developed and subsequently simplified for in vitro maturation and in vitro fertilization (IVF) of bovine follicular oocytes obtained from abattoir ovaries. After in vitro fertilization, successful cleavage and in vitro development were obtained using a simple and efficient cumulus cell co-culture method, which consistently produced 45 to 50% morulae during the treatment and culture of over 6000 bovine oocytes. Embryonic cell number for IVF-derived embryos was monitored at different stages of in vitro development during cumulus cell co-culture to evaluate embryo growth and assess embryo quality.     

Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes

BACKGROUND: The purpose of this study was to determine if there is any additional benefit from FSH priming in addition to hCG priming on in-vitro maturation (IVM) programmes. METHODS: Sixty women with polycystic ovary syndrome (PCOS) who underwent 68 IVM cycles were randomized by computer-generated numbers to receive FSH stimulation or not. Thirty-five cycles were pretreated with 75 IU rFSH for 6 days, and 33 cycles were not. Every cycle was given hCG 10,000 IU 36 h before oocyte retrieval. Immature oocytes were matured in vitro and fertilized by ICSI, and the resulting embryos were replaced on day 2 or 3. RESULTS: A total of 1528 immature oocytes were recovered. The overall maturation and fertilization rates were 74.2 and 72.8% respectively. After embryo transfer, 23 pregnancies resulted (33.8%). The oocyte numbers and endometrial thickness were similar between FSH-primed and non-FSH-primed groups. Serum estradiol level on the day of hCG injection was significantly higher in the FSH-primed group than in the non-FSH-primed group (377.2 pmol/l versus 143.8 pmol/l, P=0.001). The maturation rate, fertilization rate and pregnancy rate were 76.5, 75.8 and 31.4% respectively for FSH-primed group, and 71.9, 69.5 and 36.4% respectively for non-FSH-primed group (all not significant). CONCLUSIONS: IVM is a feasible treatment for women with PCOS. FSH priming has no additional beneficial effect on IVM.     

Cytogenetic analysis of human oocytes and embryos in an in-vitro fertilization programme.

Oocytes (unfertilized and preovulatory) and embryos (normal and polypronuclear), which were donated to research by patients undergoing procedures of assisted reproductive treatment, were analysed for cytogenetic abnormalities. A total of 362 oocytes and embryos were analysed. The unfertilized oocytes with readable metaphases (53.4%) gave 25.2% chromosomal abnormality with diploidy being the main aberration observed. A high incidence of premature chromosome condensation (PCC) was observed and the incidence of PCC in oocytes exposed to colcemid was significantly higher (14/62, 22.6%) than in those not exposed to this treatment (3/41, 7.3%, P less than 0.05). When chromosomal anomalies and PCC in the unfertilized oocytes were correlated to various patient criteria such as stimulation regimen, number of human menopausal gonadotrophin ampoules, peak oestradiol levels, age of patient and number of previous attempts, none of the criteria tested had any significant relationship to the incidence of chromosomal abnormality. However a significant increase in the incidence of PCC was noted in the gonadotrophin-releasing hormone (GnRH) 'flare' group (6/15, 40.0%) compared to the GnRH 'down-regulation' group (11/88, 12.5%). The incidence of chromosomal abnormalities among preovulatory oocytes was 16.7% and diploidy was the only abnormality noted. For embryos arising from two-pronuclear oocytes, the chromosomal constitution related mainly to embryo quality. The rate of chromosomal abnormality for apparently good quality embryos was 23.5% and for poor or fragmented embryos 83.3%. The majority (77.3%) of the readable metaphase plates for polypronuclear 1-cell and cleaved embryos showed grossly abnormal chromosome complements but 19% of the cleaved embryos contained sets of normal diploid chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Andrology: Subzonal sperm insertion with aged human oocytes from failed in-vitro fertilization attempts: fertilization results and some applications

This study was undertaken to assess the potential of aged humanoocytes from failed in-vitro fertilization attempts as a modelfor the study of fertilization events after subzonal sperm insertion(SUZI). Criteria of aged oocyte suitability for this purposewere (i) the absence of nuclei, (ii) the presence of a polarbody, (iii) the absence of cell division or fragmentation, (iv)marked ooplasmic contraction in hyperosmotic medium, and (v)rapid ooplasmic relaxation after returning into normo-osmoticmedium following the micromanipulation. Micro-injection techniqueswere essentially the same as for SUZI with fresh oocytes. Oocytesthat fused with the micro-injected spermatozoa developed pronucleiof typical internal structure. However, the number of pronucleiwas often higher than that theoretically expected if each spermnucleus incorporated into the oocyte gave rise to a single pronucleus.Thus, the use of aged oocytes implies the need for a specificmethod to assess the frequency of fusion in sperm samples examined.The results suggest that the method described here can be appliedin a preliminary diagnostic test before a therapeutic SUZI attemptand in studies aimed at the optimization of sperm treatmentprotocols to increase the fusion capacity of subzonally insertedspermatozoa.     

未成熟卵母细胞在TCM199与P1培养体系体外成熟的比较

目的 探讨人未成熟卵母细胞适宜的培养体系及培养时间.方法 将卵母细胞-卵丘复合体(0CC)332枚和裸卵(DO)393枚随机置入TCM199和P1两种培养体系体外成熟.OCC体外培养48 h,观察其成熟情况.DO分别在24、30、48 h观察第1极体排出情况,计算体外成熟率.OCC和DO成熟后称为成熟的卵母细胞,成熟的卵母细胞行精子卵浆内注射技术(ICSI)受精,比较其在两种培养体系的受精率、卵裂率及囊胚形成率.结果 在TCM199和P1培养体系中,OCC的成熟率、成熟后成为成熟的卵母细胞的受精率和卵裂率的差异无统计学意义(P均>0.05),但成熟的卵母细胞的囊胚形成率为53.7%比37.8%(P<0.05).DO在TCM199和P1培养体系中体外培养24、30、48 h时成熟率的差异无统计学意义;由DO成熟后形成的成熟的卵母细胞在P1培养体系中的受精率、卵裂率和囊胚形成率高于其在TCM199培养体系中,但只有受精率差异具有统计学意义(73.5%比62.9%,P<0.05);DO在两种培养体系中体外培养48 h和30 h的成熟率均明显高于24 h(P<0.05),但48 h与30 h的成熟率相比无差异.结论 OCC适宜在TCM199培养体系的培养;DO适官在P1培养体系的培养.DO体外成熟形成成熟的卵母细胞的时间可以缩短至30 h.     

Micromorphometry and spermatozoa binding patterns of fertilized and unfertilized human oocytes after in-vitro fertilization

Human oocytes (n = 380) from 71 in-vitro fertilization patientswere measured 18 h after insemination to find out if certainparameters of oocyte morphology could be related to fertilization.In addition, the number and distribution patterns of spermatozoabound to or within the zona pellucida of 534 oocytes were analysed.The mean diameter of the human oocyte was 167.7 ± 9.5µm and its mean volume was 2.5 x 10⁶ µm³. Therewere no significant differences in diameter between 112 fertilizedand 168 unfertilized oocytes, although they displayed differencesin the size of the perivitelline space and the zona pellucida.The age of the patients had no significant effect on the morphometryof the oocytes. Sperm binding patterns did not correlate withfertilization. The number and distribution of the spermatozoaon the surface of the zona pellucida was extremely heterogeneousand was not related to the occurrence of fertilization. Allpossible binding and distribution patterns were in the samerange in both fertilized and unfertilized oocytes. In conclusion,the micromorphometry of human oocytes and their sperm bindingpatterns were not related to the occurrence of fertilization.