The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization.

Cryopreservation of mouse oocytes induced a high rate of atresia. Frozen oocytes observed immediately after thawing did not exhibit any alteration in the frequency of chromosomal abnormalities, aneuploidy or polyploidy. After in-vitro fertilization attempts, the cleavage rate of frozen-thawed mouse oocytes was decreased. Cytogenetical observations of inseminated eggs also confirmed this decrease in fertilization rate. First and second cleavages were delayed compared to fresh controls but subsequent development to the 4-cell stage was not altered. Freeze-thawing increased the incidence of chromosomal abnormalities in inseminated oocytes but this only concerned the frequency of triploidy and not monosomic or trisomic aneuploidy. The increase in triploidy seemed to be largely due to the presence of digynic embryos. Second polar body retention seemed to be mainly responsible for this high rate of polyploidy.     

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.     

Cytogenetic analysis of human oocytes from fertile women.

Several reports have shown a relatively high incidence of chromosome anomalies in human inseminated-unfertilized oocytes from infertile women. In the present study, cytogenetic analysis was attempted in 73 human oocytes from 17 fertile women in order to establish the incidence of chromosome anomalies in the fertile population and to compare this with the incidence in inseminated-unfertilized oocytes from infertile women. Of 56 oocytes that could be analysed, 42 oocytes were haploid, 12 were hypohaploid and two exhibited fragmented chromosomes. The low rate of chromosome anomalies (3.6%) found in this population suggests that there is natural selection at fertilization against diploid oocytes and oocytes with fragmented chromosomes. This result also questions the high incidence of chromosome anomalies found in previous reports using inseminated-unfertilized oocytes.     

Fertilization and early embryology: Granulosa cells improve human embryo development in vitro

A total of 17 couples with repetitive implantation failure aftertransfer of fresh or frozen—thawed embryos had half oftheir zygotes cultured in standard conditions and frozen atday 2 after insemination, and the other half cocultured withautologous granulosa cells and transferred at the morula orblastocyst stage at day 5 or 6 after oocyte retrieval. At theend of the culture period, supernatants of cocultures were recoveredfor steroid assays. Monolayers were stained for granulosa cellgrowth and morphological assessment. We observed that granulosacells improve embryo development in vitro since 32 out of 60(53%) reached the morula stage and 18 (30%) the blastocyst stage,leading to a total of 83% embryos available for transfer (comparedwith 3% without coculture). The ongoing pregnancy rate of thesepatients who were selected because they had at least three previousimplantation failures, is only 5.9%, however, which is similarto the control group without coculture (6.3%). To conclude,granulosa cells improve embryo development but not the pregnancyrate after transfer of cocultured embryos in patients with multipleprevious implantation failures.     

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.     

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.     

Cryopreservation of human embryos and oocytes

The success rate of human embryo cryopreservatlon depends ontechnical and embryonic parameters. First of all, the cryoprotectantcan affect embryo survival as we found by comparing two freeze-thawprocedures using propanediol (PROH) (1.5 mol) alone or withsucrose (0.1 mol). Embryo survival was significantly enhancedwith sucrose (62 versus 32%). Embryo quality is another majorparameter involved in the success of freezing; the rates ofpositive survival were found to be 67% for morphologically normalembryos versus 49% for embryos with fragments (P < 0.001).The efficiency of embryo cryopreservatlon in an IVF programmecould be estimated in 1986: a woman with extra embryos, storedafter transfer of 3–4 fresh embryos (16% of all cydes),can expect a 22% pregnancy rate per transfer of fresh embryosand a 32% pregnancy rate per collection after transfer of thestored eggs. A comparative study of the cryopreservability ofimmature or mature oocytes was performed in humans. Human oocyteshave a low survival rate (36%) whatever the cryopreservationprotocol or the initial maturation stage. Immature human oocytescould survive freezing and thawing, mature and be fertilizedin vitro, but with a very low efficiency.     

Embryo development and pregnancies from in-vitro matured and fertilized human oocytes.

There is an increasing interest in retrieving immature oocytes in the absence of or with limited gonadotrophin exposure, with the aim of maturing them in vitro for embryo transfer purposes. The aim of this report is to present our experience of fertilization, embryonic development and pregnancies from in-vitro maturation cycles. A total of 18 patients underwent 21 cycles in which an average of 8.1 immature oocytes was retrieved after limited exposure to human menopausal gonadotrophin (HMG) and no exposure to human chorionic gonadotrophin (HCG). In one cycle, no oocytes were recovered. The oocytes were cultured for 44 h and 121 oocytes which reached MII were injected with a single spermatozoon. A total of 71 oocytes showed two pronuclei and 53 zygotes cleaved. Forty-four embryos were transferred in 17 cycles. Five weeks after embryo transfer, ultrasound examination indicated the presence of one gestational sac and one fetal heart beat in two patients. The results suggest that in-vitro matured oocytes can undergo fertilization and the resulting embryos may result in pregnancies. However, the success rate was not sufficient to recommend widespread use of the technique without further research.     

Fertilization in vitro of human oocytes by spermatozoa collected in different stressful situations

It has been shown that semen quality is impaired in couplesundergoing in-vitro fertilization (IVF), probably due to stress.A possible effect of stress on the ability of spermatozoa tofertilize human oocytes in vitro was analysed in the presentstudy composed of 26 couples with normozoospermic men undergoingIVF. A semen sample was obtained during the infertility work-upand was cryopreserved (sample 1). A second sample (sample 2)was provided after oocyte retrieval during the IVF cycle. Sample1 was thawed and both samples were washed and preincubated foroocyte insemination. One-hundred-and-five oocytes were inseminatedusing thawed sample 1, and 120 with sample 2.Semen parameterssuch as density, progressive motility and percentage of abnormalforms were compared between sample 1, before and after freezing,and sample 2. Only motility was significantly (P<0.01) decreasedby cryopreservation in sample 1, but no parameter was significantlydifferent when fresh sample 1 was compared to sample 2. Thefertilization rate was 78.6% using sample 1 in comparison to87.5% when sample 2 was employed (not significant, NS). Cleavagerates were 77.7 and 89.7%, respectively (NS). A group of fivepatients undergoing IVF who needed donor semen served as a controlfor the effect of sperm cryopreservation on IVF. In these cases,the donor was asked to provide a fresh sample. Half of thissample was frozen and thawed. Subsequently, fresh and thawedsamples were prepared for insemination and oocytes inseminatedeither with the fresh preparation (n=24) or the frozen and thawedspermatozoa (n=22). There was a significant (P<0.05) decreasein motility in the thawed sample, but fertilization and cleavagerates were not different. These data suggest that the stressfulsituation induced by IVF treatment in normozoospermic men doesnot affect the ability of spermatozoa to fertilize human oocytesin vitro. Cryopreservation of human spermatozoa before IVF maybe a good policy in couples especially suspected of being understress during this procedure.     

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro.

It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.     

Fertilization and early embryology: Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection

The behaviour of sperm cells after intracytoplasmic sperm injection(ICSI) was investigated by analysing 192 unfertilized and 37one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilizedoocytes were directly fixed whereas 110 were first parthenogeneticallyactivated by puromycin. In contrast to the findings in unfertilizedoocytes after in-vitro fertilization, most unfertilized oocytesafter ICSI (n = 76) contained evidence of the presence of spermatozoain the cytoplasm. Few oocytes (n = 6) contained prematurelycondensed sperm chromosomes (PCC), whereas the majority containedeither intact sperm heads (n = 31) or swollen sperm nuclei (n= 39) along with metaphase II chromosomes of the oocyte. Followingactivation by puromycin, swollen sperm nuclei and PCC were nolonger observed, whereas unchanged sperm heads persisted in12 oocytes displaying a single pronucleus. A non-decondensedsperm nucleus along with decondensed maternal chromatin werealso discovered in 32 out of 37 oocytes displaying a singlepronucleus after ICSI. The findings in unfertilized and 1PNoocytes after ICSI indicate that successful sperm injection,even followed by oocyte activation, is not sufficient to guaranteenormal fertilization. It seems that partial sperm membrane damageprior to injection is also required to ensure normal sperm decondensation.     

The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation.

A prospective randomised study was performed to evaluate stimulated versus natural oviductal environment in comparison with in-vitro culture for the developmental capacity of mouse embryos. Therefore, embryos of superovulated F1 hybrid CBAxC57Bl females were collected at 17, 22, 41 and 46 h after human chorionic gonadotrophin treatment and randomly divided into five groups. They were either transferred immediately to untreated pseudopregnant females, cultured in vitro for 5, 24 or 29 h before transfer, or cultured in vitro for 96 h to blastocysts. The transfers resulted in an impaired implantation (P < 0.001) and a lower numbers of living fetuses (P < 0.001) when embryos had been exposed longer to the stimulated oviductal environment. Similar results were obtained after a longer period of in-vitro culture (P < 0.05). However when embryos were flushed earlier from the superovulated mice and cultured longer in-vitro until the transfer was performed, the implantation rate was improved (P < 0.01). Blastocyst development, however, was better (P < 0.001) when embryos were flushed later. In conclusion, the stimulated oviductal environment impairs the developmental capacity of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated oviductal environment.     

Regulators of sperm function: Composition of the human zona pellucida and modifications following fertilization

The composition of individual human zonae pellucidae and modificationsto this extracellular coat both before and after fertilizationwere analysed using a rapid, sensitive, non-radioactive biotinylation-or lectin-based detection system; these assays use commerciallyavailable reagents and can be performed on fragments of individualzonae pellucidae. The zona pellucida from unfertilized eggsis composed of three glycoprotein species designated as huZP1,huZP2 and huZP3. Under non-reducing conditions, the molecularweights of these proteins are 150 kDa, 100 kDa, and 55–65kDa respectively. Following fertilization, huZP1 was not detectedunder either non-reducing or reducing conditions. In contrast,after fertilization huZP2 was detected under non-reducing conditions,but not under reducing conditions. The ability to detect pre-and postfertilization changes in a single human zona pellucidais discussed in relation to its value in assessing deficienciesin clinical and laboratory protocols used for in-vitro fertilization.     

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.     

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.     

Oocyte dysmorphism and aneuploidy in meiotically mature human oocytes after ovarian stimulation.

The frequency of aneuploidy in 583 newly aspirated, uninseminated metaphase II-stage human oocytes which exhibited seven distinct forms of cytoplasmic dysmorphism [Van Blerkom (1990) J. Electron Microsc. Tech., 16,324] after ovarian stimulation and ovulation induction was determined in the living state by DNA fluorescence followed by fixation and air-drying for karyotyping. The findings demonstrate that as many as half of the oocytes with dysmorphic phenotypes which arise early in meiotic maturation are aneuploid, with hypohaplidy predominant. In contrast, cytoplasmic defects which occur at or after metaphase I are associated with a relatively low frequency of aneuploidy (less than 15%), which is comparable to that previously reported for human oocytes with a normal cytoplasmic appearance [Van Blerkom and Henry (1988) Hum. Reprod., 3, 777]. The aetiologies of aneuploidy in dysmorphic oocytes, as well as the clinical implications for oocyte selection in laboratory-assisted conception are discussed.     

EndocrinologyHormonal profile during the follicular phase in cycles stimulated with a combination of human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonist (Cetrorelix)

A third-generation gonadotrophin-releasing hormone antagonist(Cetrorelix) was used during ovarian stimulation in 32 patientsundergoing assisted reproduction, in order to prevent the prematureluteinizing hormone (LH) surge. In all patients, ovarian stimulationwas carried out with two or three ampoules of human menopausalgonadotrophin (HMG), starting on day 2 of the menstrual cycle.In addition, 0.5 mg of Cetrorelix was administered daily fromday 6 of HMG treatment until the day of ovulation inductionby human chorionic gonadotrophin (HCG). A significant drop inplasma LH concentration was observed within a few hours of thefirst administration of Cetrorelix (P<0.005). Moreover, noLH surge was detected at any point in the treatment period inany of the 32 patients. A mean oestradiol concentration of 2122±935ng/1 was observed on the day of the HCG administration, indicatingnormal folliculogenesis. Like LH, progesterone concentrationalso dropped within a few hours of the first administrationof Cetrorelix (P< 0.005). A 0.5 mg daily dose of Cetrorelixprevented a premature LH surge in all the 32 patients treated.     

Preliminary findings in germinal vesicle transplantation of immature human oocytes

Transplanting a germinal vesicle (GV) from an aged woman's oocyte into a younger ooplasm has been proposed as a possible way to reduce the incidence of oocyte aneuploidy which is considered to be responsible for age-related infertility. In this study, we have assessed the efficiency of each step involved in nuclear transplantation-specifically cell survival, nuclear-cytoplasmic reconstitution, and the capacity of the reconstituted oocytes for in-vitro maturation. In addition, we have evaluated the fertilizability and karyotypic status of the manipulated oocytes by intracytoplasmic sperm injection (ICSI) and fluorescent in-situ hybridization technique respectively. Nuclear transplantation was accomplished with an overall efficiency of 73%. Due to the limited availability of materials, most nuclear transplantation procedures were performed between sibling oocytes. The maturation rate of 62% following reconstitution was comparable with that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The ICSI results of the reconstituted oocytes yielded a survival rate of 77%, a fertilization rate of 52%, and a satisfactory early embryonic cleavage. Furthermore, in a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, there was an appropriate chromosomal segregation. These findings demonstrate that human oocytes reconstituted with GV nuclei are able to undergo maturation, fertilization, and early embryo cleavage, and maintain a normal ploidy. Although in-vitro maturation seems to be a limiting step, this technique would allow us to investigate further the nuclear-ooplasmic relationship during meiotic maturation.     

The effect of preinsemination interval upon fertilization of human oocytes in vitro

A correlation between oocyte maturity, duration of preinseminationinterval (PII) and fertilization rate in vitro has been suggested.Therefore, delayed insemination is being widely practised inmany IVF centres. The purpose of this study was to investigateprospectively the effect of PII upon the fertilization ratein vitro. A total of 1474 oocytes were inseminated followingincubation for between 30 and 540 min. The duration of the PIIwas determined randomly. It was found that neither fertilizationrate (on average 46.9%) nor pregnancy rate (20.4%, calculatedper embryo transfer) were affected by PII for any given degreeof oocyte-cumulus maturity. The ability to inseminate oocytesat any time (within 9 h following egg collection) to the convenienceof the biologist further simplifies the technology of IVF.     

Fertilization and early embryology: Morphometric characterization of normal and abnormal human zygotes

Human zygotes (n = 278) from 96 in-vitro fertilization gonadotrophin-stimulatedcycles were photographed in their pronuclear stage (16–18h post-insemination). Normal morphological fertilization (twopronuclei) was observed in 215 zygotes, 17 showed only one pronucleus,40 showed three pronuclei and six showed four. Area, perimeterand maximum and minimum diameters of each zygote and pronucleuswere measured using an IBAS 2000 (Kontron) image analyser. Whenthe four groups were compared, whole zygotes did not show anymorphometric difference. However, pronuclei from these groupsshowed that a high number of pronuclei was directly relatedto small pronuclei. Differences in pronuclear size and a linearincrease in the nucleus/cytoplasm relationship were found whichvaried with the number of pronuclei.