Fertilization and early embryology: Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos

In-vitro culture of 1-cell mouse embryos was used to assessthe influence of pentoxifylline on early embryonic development.If cultured in concentrations of 5, 10 or 50 µM, earlyembryonic development was unaffected and no differences in cellnumbers were noted in embryos reaching the blastocyst stage.However, at 3.6 and 7.2 mM, pentoxifylline inhibited cleavagefrom the 2-cell stage onwards. If 1-cell mouse embryos wereexposed for only 30 min to these concentrations, blastocystformation was found to be morphologically normal. However, cellnumbers of such blastocysts were significantly decreased afterexposure to pentoxifylline. These results may indicate thatexposure of gametes or zygotes to pentoxifylline should be avoidedas much as possible when this drug is used in human assistedreproduction. If administered at regular therapeutic doses,it is probable that no adverse effect on early embryonic developmentin vivo will occur. Further research is needed to confirm andelucidate the above findings.     

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of differentculture systems on the development of early human embryos invitro. A total of 460 fertilized oocytes from 82 cycles of patientswere transferred into one of four systems: (1) into dropletsof Ham's F10 medium+12% normal human serum (NHS); (2) co-culturedon a human granulosa monolayer; (3) co-cultured with bovineoviductal epithelial cells (BOEC); or (4) co-cultured with bovineuterine epithelial cells (BUEC). The percentage of deavage andthe morphological appearance of embryos were recorded dailyfor 72 h in each system using an inverted phase-contrast microscope.The results showed that the proportions of the fertilized oocyteswhich developed to the four-cell stage 48 h after retrievalwere, by culture system: (1) 70% (84/120); (2) 74% (85/115);(3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval,the proportions of the eight-cell stage were, by culture system:(1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70%(57/82). We conduded that a higher proportion of fertilizedoocytes developed to embryos at the eight-cell stage in systems2, 3 and 4 than in system 1. This indicates the beneficial effectof co-culture of human embryos with granulosa cell, BOEC andBUEC monolayers, which may be due to various factors.     

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.     

Characterization of epithelial cell culture from human hydrosalpinges and effects of its conditioned medium on embryo development and sperm motility

BACKGROUND: Recent studies have reported the negative impact of hydrosalpinx on IVF outcome. Toxic effects of hydrosalpinx fluid (HF) have been the main reason for the recommendation of functional surgery, salpingectomy, prior to IVF. The present study characterized hydrosalpinx epithelial cell culture and examined the effects of its conditioned medium (CM) on sperm motility, acrosome reaction and embryo development. METHODS: Normal Fallopian tubes (n = 6) and hydrosalpinges (n = 9) were used to prepare epithelial cell culture and CM. Epithelial cell characterization was confirmed using electron microscopy. Sperm motility and acrosome reaction were determined using computer-aided sperm analysis and acrobead assay respectively and embryo development by mouse embryo development assay. RESULTS: The percentage of human motile sperm incubated in hydrosalpinx CM was significantly different from those in normal Fallopian tube (NFT) CM and modified human tubal fluid medium (hTF) (control) (P < 0.05 at 3 h and P < 0.001 at 5 and 24 h), with alteration in movement characteristic, linearity, 24 h after incubation in hydrosalpinx CM (P < 0.05). However, other sperm movement characteristics remained unchanged. Reduced acrosome reaction and poor mouse embryo development were also observed in hydrosalpinx CM but not in NFT CM and hTF. CONCLUSIONS: The results suggest that hydrosalpinx epithelial cells may be producing a fluid milieu hostile to sperm and early embryo development. The established epithelial cell culture system may provide a model to further investigate the mechanisms underlying the toxic effects of HF on embryo development and the adverse effects on IVF outcomes.     

Fertilization and early embryology: Comparison of mouse embryo development in open and microdrop co-culture systems

Co-culture with numerous cell lines has been shown to improvein-vitro embryo development. It is usually performed in openculture without an oil overlay, or in relatively large volumesof medium (e.g. 0.5 ml) under oil. We compared the efficacyof open and microdrop co-culture systems using human endometrialand tuba] cell lines and mouse zygotes; Although the mean pHvalues of the media from the tubal cell cultures (both openand oil-covered) decreased significantly over 5 days of culture,this did not appear to impair embryo development Both co-cultureand microdrop culture significantly improved blas-tocyst andhatching blastocyst formation rates. The combination of thetwo techniques (microdrop and co-culture) demonstrated the highestblastocyst formation and hatching blastocyst formation rates,as well as the highest mean cell numbers in hatching blastocysts.Co-culture in a microdrop is a superior system for mouse embryoculture.     

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.     

Fertilization and early embryology: CD44 is expressed throughout pre-implantation human embryo development

The cell surface glycoprotein CD44 has been demonstrated ina variety of cell types in embryonic and adult tissues. We haveestablished that CD44 is present on human oocytes, cumulus cells,early embryos and pre-hatched blastocysts by indirect immunofluorescence.We have also shown that CD44 is present on 8–11 week placentalstroma cells, but not on the trophoblast. These findings demonstratethat CD44 is present throughout preimplantation development,and that down-regulation occurs on the embryonic surface afterimplantation.     

Fertilization and early embryology: Polypeptide profiles of human oocytes and preimplantation embryos

The polypeptides that direct fertilization and early developmentuntil activation of the embryonic genome occurs, at the 4–8cell stage in the human, are exclusively maternal in origin,and are either synthesized during oogenesis or translated laterfrom maternal mRNA. Using sodium dodecyl sulphate—polyacrylamidegel electrophoresis and silver stain, we have visualized andcompared the polypeptides present in different populations ofhuman oocytes and cleavage stage embryos obtained after superovulationand insemination in vitro. Two polypeptide patterns were resolved,differing in the region of mol. wt 69 kDa. The distributionof these patterns showed no correlation with the ability ofindividual oocytes to achieve fertilization and develop normallyto the 8-cell stage.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Fertilization and early embryology: Temporal effects of ouabain on in-vitro development of mouse zygotes

Ouabain is a specific inhibitor of Na⁺-K⁺-ATPase, an enzymewhich controls the intracellular Na⁺ and K⁺ levels. In thisstudy, in-vitro fertilized zygotes from a hybrid mouse strainwere used to examine the temporal effects of 50 µM ouabainon embryonic development in vitro during the preimplantationperiod. A higher incidence of blastocyst formation at the endof the culture period was found when embryos were cultured inthe presence of ouabain from 22 to 46 h post-insemination, orany other period that included this time period. When zygotesfrom randomly bred mice were used, inhibition of Na⁺-K⁺-ATPasewith ouabain clearly promoted development through the 2-cellblock in vitro. As Na⁺-K⁺-ATPase is the most important regulatorof intracellular electrolyte concentrations in mammalian cells,these results suggest that an ionic imbalance exists in embryoscultured in conventional media which can be positively influencedby inhibiting this enzyme.     

Fertilization and early embryology: Improved cleavage rate of human embryos cultured in antibiotic-free medium

Retarded development and blastomere fragmentation of human prelimplantationembryos represent a common phenomenon in in-vitro culture systems.Even though media composition is generally formulated to meetembryo nutritional requirements, the influence of antibioticsupplementation has not been investigated thoroughly. The presentstudy was performed to evaluate the effects of antibiotics onembryo morphology and growth in modified culture media. A totalof 196 zygotes from 18 couples was cultured in three differentmedia: (i) conventional medium (n = 99, control group); (ii)medium modified with half the standard antibiotic concentration(n = 54); and (iii) antibiotic-free medium (n = 43); 49 embryosfrom the control group were selected at the zygote stage andtransferred to the patients on day 2. The remaining 147 zygoteswere cultured to the blastocyst stage for cryopreservatlon;their morphology and cell number were assessed daily at 40,64, 88, and 112 h post-insemination. Overall cleavage rate was95% and embryo scoring revealed 91% grade 1 embryos throughoutthe culture period in the three media. Significantly highercleavage rates were obtained in the antibiotic-free medium ateach observation, including the blastocyst stage, when comparedto the other two groups. In addition, no notable improvementwas observed in the embryos cultured in a reduced concentrationof antibiotics. In conclusion, antibiotic supplementation ofmedia has an adverse effect on the growth rate of preimplantationembryos, even in reduced concentrations, suggesting that antimicrobialdrugs may interfere with the timing of cleavage events eitherby delaying or blocking embryo development.     

Fertilization and early embryology: Use of videocinematography to assess morphological qualities of conventionally cultured and cocultured embryos

Videocinematography and image analysis procedures were utilizedto evaluate the effect of conventional and coculture methodologieson morphological parameters in human embryos derived from in-vitrofertilization (IVF). Following 24–30 h of in-vitro development,cocultured embryos had more acceptable morphological featuresand less fragmentation present than embryos cultured in mediumalone. Cocultured embryos were more advanced at the time ofreplacement when compared with conventionally cultured embryos.Zona pellucida variation (20%) also occurred more frequentlyin cocultured embryos. The morphological characteristic mostenhanced after coculture was blastomere expansion. Patientswho became pregnant across both culture treatments had a higherproportion of morphologically normal embryos replaced than patientswho failed to achieve an ongoing pregnancy. Clinical pregnancyrate for patients following coculture was 49%, which was greater(P < 0.05) than the 29% detected for patients with embryosin the conventional culture group.     

Fertilization and early embryology: Diagnosis of major chromosome aneuploidies in human preimplantation embryos

A short fluorescence in-situ hybridization (FISH) procedureusing fluorochrome and digoxigenin labelled DNA probes was developedfor application in human preimplantation embryos in order toanalyse the five chromosomes most involved in human aneuploidy(X, Y, 18, 13 and 21). The chromosomes were fluorescent-stainedand detected simultaneously in 157 blastomeres from 30 humanembryos. Successful FISH analysis was achieved in 93% of theblastomeres. Aberrations for these chromosomes were found in70% of abnormally developing monospermic embryos. The majorityof normally developing monospermic embryos obtained from olderpatients were also chromosomally abnormal. By analysing allor most of the cells from these embryos, true mosaicism wasdistinguished from technique failure. Mosaic embryos, polyploidembryos with ploidies as high as 8n, haploid embryos, embryosmonosomic for 13/21 and for X, and embryos trisomic for 13/21and 18, were common in abnormally developing embryos. In contrast,aneuploidy was the main chromosome abnormality found in normallydeveloping monospermic embryos.     

Effect of culture systems on mouse early embryo development

The amount of oxygen available to embryos in drop cultures underparaffin may be limited by the gas mixture employed, the methodof equilibration or by the use of positive or negative pressurefiltration. When these factors were varied, in experiments usingSwiss outbred (SO) embryos, the only significant differencedetected was the poorer development of embryos from the 1-cellto blastocyst stage when cultured in medium without prior equilibration.Also under these conditions, the inclusion of glucose in themedium did not increase the incidence of 2-cell block, and glutaminein the presence of glucose enhanced the development of 1-cellembryos to blastocysts. One-cell (C57BL/6 x SJL) F₁ x SO embryoswere successfully cultured in HEPES buffered CZB medium withadded bicarbonate, under an atmosphere of air. The proportionof blastocysts formed in this medium, with a concentration of10 mM sodium bicarbonate, was not different from that developedin CZB under 5% CO₂ in air.     

Fertilization and early embryology: Antioxidative capacity of preimplantation embryo culture medium declines following the incubation of poor quality embryos

Total antioxidative capacity (TAC), a measure of overall free-radicalscavenging potential, was determined by enhanced chemiluminescencein preimplantation embryo culture medium (PECM; pre-equilibratedHam's F-10 medium supplemented with 7.5% patient's serum). Changeswere evaluated in PECM TAC following a 24 h incubation of 66single human embryos, as was TAC of patient's serum alone. ThePECM TAC averaged 8.1% of the same patient's blood serum TAC.The percentage decline of PECM TAC over an incubation periodof 24 h ranged from 0.9 to 41.7%, with a median of 5.5%. Thedecline in PECM TAC in different embryo quality groups was alsostudied. Embryos were categorized as ‘good’, ‘fair’or ‘poor’ according to a scoring system based onan assessment of both the morphological appearance and developmentalspeed of the embryos. Incubation of poor quality embryos wasassociated with a decline in TAC, which was significantly higherthan that observed in ‘good’ and ‘fair’embryos. The findings suggest that impaired embryo developmentmay be associated with an increased generation of reactive oxygenspecies by the embryo.     

Fertilization and early embryology: The effect of pentoxifylline on mouse in-vitro fertilization and early embryonic development

This study was designed to assess the effect of pentoxifylline(PTX) on fertilization and early embryonic development in themouse. Oocytes from superovulated B6CBA female mice were inseminatedin vitro with spermatozoa from B6CBA males incubated with PTXaccording to different protocols, i.e. (i) 3.6 and 7.2 mM PTXwashed out prior to insemination, (ii) 3.6 and 7.2 mM PTX dilutedsix times in the insemination medium and (iii) PTX present at3.6 and 7.2 mM in the insemination medium. After inseminationand washing, fertilization was assessed by the presence of 2-cellstage embryos. These were further cultured up to the blastocystor egg-cylinder stage to asses embryonic development. Parthenogeneticactivation was evaluated by exposing post-ovulatory oocytesto 3.6 and 7.2 mM PTX. If spermatozoa were washed free fromPTX before insemination, no effect on either fertilization orsubsequent development was found. If PTX was not washed out,fertilization was reduced significantly, yet development offertilized oocytes was unaffected. If insemination was performedin the presence of PTX both fertilization and development wereimpaired. Parthenogenetic activation was not increased by PTXexposure. We conclude that if used in in-vitro fertilization,exposure of oocytes and/or zygotes to PTX has to be avoidedby washing out the compound thoroughly to prevent adverse effectson early embryonic development.     

Fertilization and early embryology: Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium

The formulation of chemically defined culture media that supportprimate embryo development would facilitate studies on primatepreimplantation embryogenesis. The specific aims of this studywere (I) to evaluate the development of macaque embryos in asimple, chemically defined, protein-free medium developed fora rodent embryo model, and (ii) to determine if a two-step progressiveculture system could enhance blastocyst development and zonaescape. In experiment 1, in-vitro-fertilized pronucleate stageembryos (n=81) from nine monkeys were randomly allocated toone of three treatments: (a) hamster embryo culture medium-6(HECM-6; chemically defined, protein- free medium), (b) CMRL-BCSmedium (modified CMRL 106 medium containing 20% bovine callserum; BCS), and (c) a two-step culture procedure (HECM-6 throughto the 8- to 12-cell stage, and CMRL-BCS medium beyond thatstage). Optimal development was attained equally (P0.05) withembryos cultured in CMRL-BCS medium or the two-step procedure(48 and 61% blastocysts respect ively). HECM-6 alone supporteddevelopment to the morula stage (72%) equally as well as CMRL-BCSmedium (80%) or the two-step procedure (69%), but not to theblastocyst stage (22 versus 48 and 61% respectively). Hatchingof the blastocysts was essentially limited to the serum-containingmedia (CMRL-BCS medium, 31% two-step procedure, 44%). In experiment2, in-vitro-fertilized pronucleate-stage embryos (n=87) fromnine monkeys were randomly placed in each of four two-step treatments:(a) HECM-6 through to the 8- to 12-cell stage and CMIRL-BCSmedium beyond that stage, (b) HIECM-6 through to the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HIECM 6 throughto the morula stage and CMIRL-BCS medium beyond that stage,and (d) HECM-6 through to the morula stage and HECM-6-BCS beyondthat stage. Greater (P 0.05) percentages of embryos developedinto blastocysts, expanded blastocysts and hatched blastocystswhen switched at the 8- to 12-cell versus the morula stage inthe second step medium. When transferred into BCS containingmedium at either the 8- to 12-cell or morula stage, embryosunderwent blastulation and expansion equally well in CMIRL-BCSmedium versus HECM-6-BCS. However, when embryos were switchedto the second step medium at the 8- to 12-cell stage, hatchedblastocysts 1690 were obtained more (P0.05) frequently in CMRL-BCSmedium (50.9%) than in HECM-6-BCS (37%). This work is the firstto produce in-vitro-fertilized primate blastocysts culturedfrom the pronucleate stage in chemically defined, protein-freemedium, and demonstrates that while primate embryos can formmorulae in such a medium, their requireents for blastocoel formationand zona escape appear to be more demanding, and may be acquiredas early as the 8-cell stag.     

Fertilization and early embryology: Sexual differentiation and preimplantation cell growth

Growth rates of human preimplantation embryos fertilized invitro were assessed and compared to the sex of the pregnancyoutcome. The likelihood of a liveborn male was significantlygreater than that of a female if the mean number of cells/embryowas four or greater at the scheduled time of transfer (oddsratio of 6: 1). This finding suggested that the Y chromosomeexpresses factors which influence embryonic growth rates immediatelyafter fertilization.     

Fertilization and early embryology: The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme

The cumulative embryo score system involves three aspects ofrelevance in pregnancy achievement during in-vitro fertilization(IVF) and embryo transfer: cleavage rates, morphological qualitiesand the number of embryos transferred. The scores of 602 IVF/embryotransfer trials were calculated and analysed to determine thesystem's relationship to pregnancy rate, pregnancy outcome andthe incidence of twin and triplet pregnancies. The system wasalso applied to cycles where endotoxins were either presentin or absent from culture medium, in order to evaluate its validityin quality control analyses. Pregnancy rates were found to increasefrom 4%, with scores between 1 and 10, to 35% in the 41–50group. The score of 20 was the criterion for separating patientsinto poor and good pregnancy prognosis groups (P = 0.00001).Biochemical abortions occurred more frequently with scores <20(P = 0.00978), but a similar relationship was not found in clinicalabortion rates (P = 0.62206). Birth rates below and above ascore of 20 (2.8 and 19.2%, respectively) differed significantly(P = 0.0005). The scores of twins overlapped extensively withthose of singleton births, but those of all triplets were >40. The system did not reflect a correlation between embryoquality and the presence of endotoxins in culture medium.     

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro

The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.