The influence of bacterial vaginosis on in-vitro fertilization and embryo implantation during assisted reproduction treatment.

There is growing evidence that the pathogenic effects of bacterial vaginosis may not be confined to the lower genital tract. Possible associations with infertility and effects on fertilization and implantation were studied in patients undergoing in-vitro fertilization (IVF) treatment. High vaginal swabs taken at the time of oocyte collection were assessed by Gram staining. The prevalence of bacterial vaginosis and of intermediate and normal flora in 301 patients was 25.6, 14.0 and 60.4% respectively. Bacterial vaginosis was more prevalent in patients with tubal (31.5%, n = 149) compared with non-tubal (19.7%, n = 152) infertility (odds ratio (OR) 1.87, CI 1.11-3.18, P = 0.02). Bacterial vaginosis did not have an adverse effect on fertilization rate. Further, no significant difference in implantation rates was seen when comparing bacterial vaginosis (15. 8%, OR 1.03, CI 0.66-1.61) and intermediate flora (13.1%, OR 0.82, CI 0.45-1.52) with normal flora (15.5%). Though confidence intervals around the observations were relatively wide, the findings suggest that routine screening for bacterial vaginosis in the hope of improving the success of IVF treatment is not justified. The prevention of complications in pregnancy associated with bacterial vaginosis might be a more relevant indication for screening at the time of IVF treatment, in particular patients with tubal disease, if treatment were shown to be effective for that particular purpose. However, antibiotic treatment before IVF has been shown to be positively disadvantageous for IVF by encouraging other organisms.     

Effect of retinoic acid on implantation and post-implantation development of mouse embryos in vitro

BACKGROUND: This study was designed to examine the embryotoxic potential of retinoic acid (RA) at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. METHODS AND RESULTS: All-trans retinoic acid (t-RA) was administered to ICR mice embryos at a dose level of 0, 0.001 micromol/l, 0.1 micromol/l and 10 micromol/l throughout in-vitro culture. A total of 404 embryos was randomly assigned to all different dose groups. The percentage of embryos in later stages of development changed depending upon the dose of RA used. Exposure to 10 micromol/l of t-RA at the blastocyst stage, implanted blastocyst stage or early oocyte stage was also found to cause different degrees of retardation of embryo development and embryo death. These findings demonstrate, for the first time, that RA exerts an adverse effect on embryo growth during the early post-implantation stages of development, in comparison with day 3 to day 8 of gestation in vivo. CONCLUSIONS: Although isotretinoin (13-cis-retinoic acid) is effective for the therapy of cystic acne and other dermatological disorders, retinoid treatment should be avoided at the early post-implantation stage of gestation.     

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.     

Pregnancy: Effect of pentoxifylline on implantation and post-implantation development of mouse embryos in vitro

Previous work from our laboratory has revealed that brief exposureof mouse zygotes to 3.6 and 7.2 mM pentoxifylline reduced cellnumbers in mouse embryos reaching the blastocyst stage in vitro.Since a decrease in cell numbers may impair further development,the present study aimed to assess the implantation in vitroand the development beyond implantation of these embryos. Blastocystscultured from the one-cell stage in the presence of 5, 10 and50 µM pentoxifylline, i.e. concentrations expected inthe secretions of the genital tract after oral administration,were shown to develop normally to the egg cylinder stage invitro. Blastocysts, obtained after culture in vitro of zygotesexposed 29 h after human chorionic gonadotrophin (HCG) for 30min to 3.6 and 7.2 mM pentoxifylline, i.e. the concentrationsused for enhancing sperm function in vitro, could implant buttheir development to the egg cylinder stage was found to beimpaired. The same effect was noticed if blastocysts, grownin vitro from the one-cell stage, were exposed to 3.6 and 7.2mM pentoxifylline for 30 min in vitro 120 h after HCG. Thesefindings imply that when pentoxifylline is used to enhance spermfunction in vitro, it should be washed out of the preparationused for insemination.     

Effect of preantral follicle isolation technique on in-vitro follicular growth,oocyte maturation and embryo development in mice

BACKGROUND: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. METHODS: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. RESULTS: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. CONCLUSIONS: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.     

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.     

Implantation: Effect of platelet activating factor on embryonic development and implantation in the mouse

Platelet activating factor (PAF) was administered to femalemice in order to investigate its effect on ovulation rate andon oocyte quality including their in-vitro embryonic development,implantation and uterine receptivity. In experiment 1, 4-week-oldfemale mice were assigned to receive PAF or phosphate bufferedsaline for 4 consecutive days. On the second day of this treatment,pregnant mares' serum gonadotrophin was administered and humanchorionic gonadotrophin (HCG) 48 h later, after which copulationoccurred. Oocytes were collected on the following day and evaluated.The mean number of oocytes and zygotes (two pronuclear stageembryos) recovered from the PAF-treated group was not differentfrom the control group (31 versus 27), but the proportion ofzygotes was higher in PAF-treated group than in controls (83versus 68%, P 0.05, PAF versus controls). Although the rateof in-vitro first cleavage was not different in the two groups(82 versus 69% respectively), hatching was higher in the PAF-treatedgroup than control mice (99 versus 83%, P 0.01). In experiment2, the in-vitro developed blastocysts from experiment 1 weretransferred into the uterus of day 3 pseudopregnant PAF-treatedor control recipients. Three different combinations of intrauterinetransfer were performed; PAF embryo to control recipient (PAFC:n = 19), control embryo to PAF recipient (CPAF: n = 19), andcontrol embryo to control recipient (CC: n = 22). Implantationand abortion were assessed on day 19 post-transfer. The implantationrate of CPAF (23.7%) was lower than CC (31.1%, P 0.05), butwas not different from PAFC (31.2%). Further, CPAF showed ahigher abortion rate per embryo (29.6%) than PAFC (12.7%, P 0.05), but was not different from CC (24.4%). In the presentstudy, PAF administration enables females to produce oocyteswith a higher potential for fertilization, in-vitro developmentand implantation, but has a detrimental effect on uterine receptivityto embryos.     

Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells

A study was undertaken to evaluate embryonic development andestablish pregnancies with human embryos after in-vitro culturein two different systems. Treatment A consisted of culturingzygotes in serum-supplemented human tubal fluid culture medium(HTF). Treatment B consisted of culturing zygotes on a monolayerof bovine oviductal epithelial cells with HTF. At the time ofembryo replacement, embryos in treatment B had 4.11 blastomerespresent, which was greater (P < 0.05) than the 3.81 presentfor embryos in treatment A. In addition, the cellular fragmentationrate for treatment A embryos was 1.10, which was greater (P< 0.05) than the fragmentation rate of 0.38 for embryos withintreatment B. The incidence of ongoing pregnancy was higher afterreplacement of co-cultured embryos (treatment B) (43%) thanreplacement of conventionally cultured embryos (treatment A)(29%). The implantation rate per embryo increased (P < 0.05)from 11.5 to 18.4% after co-culture. In treatment B the proportionof ‘spare’ embryos developing to expanded blastocystswas 58.5%, which was greater (P < 0.05) than the blastocystdevelopment rate of 29.3% observed for embryos within treatmentA.     

In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells

Human zygotes resulting from IVF were placed in two differentculture systems to evaluate in-vitro development and to establishpregnancies in patients following embryo replacement. TreatmentA (control) consisted of culturing zygotes in a modified Earle'sBalanced Salt solution while treatment B consisted of culturingzygotes on a monolayer of fetal bovine uterine fibroblasts inthis same culture medium. At the time of embryo replacement,embryos within treatments A and B had 3.7 and 4.3 blastomerespresent, respectively. After 24 h in culture, the cellular fragmentationrate for treatment A embryos was 0.85 which was greater (P <0.05)than the fragmentation rate of 0.44) for embryos within treatmentB. The incidence of implantation for patients whose embryoswere given treatment A was 17.0% which was lower (P <0.05)than 35% for those given treatment B. Implantation rates increasedwith time in culture (43%) for treatment B embryos. Cultureby treatment B of three pronucleate zygotes resulted in 7/9and 4/9 reachIng the blastocyst and expanded blastocyst stages,respectively, whereas only 1/26 three-pronucleate zygotes culturedusing treatment A reached either of these stages.     

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: 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: 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: 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.     

Cigarette smoking and outcomes of in-vitro fertilization and embryo transfer: a prospective cohort study.

A prospective cohort study of 222 consecutive couples undergoing 297 cycles of in-vitro fertilization and embryo transfer (IVF-ET) was conducted to evaluate the impact of cigarette smoking in males and females. Compared with non-smokers, females smoking at the time of treatment had more previous pregnancies (1.16 versus 0.63, P less than 0.001), consumed more coffee per day (3.29 versus 1.85 cups, P = 0.001) and were less likely to hold a professional or skilled job (41% versus 66%). There was no difference in the response to ovarian stimulation in terms of the duration and dose of human menopausal gonadotrophin, peak oestradiol level or number of oocytes retrieved. The fertilization rate was actually higher in heavy smokers than in non-smokers (79.3% versus 61.3%, P = 0.007). The rate of embryo cleavage was retarded in a dose-dependent fashion. In smokers of 1-14 cigarettes/day, the likelihood of transferring an embryo at greater than or equal to 4-cell stage was 0.87 [95% confidence limits (CL) 0.56-1.4] and in smokers of greater than or equal to 15 cigarettes/day, the likelihood was 0.52 (95% CL 0.31-0.88). However, evaluation of interrelated factors using logistic regression suggested that a low socioeconomic status had a greater detrimental effect on embryo cleavage rate than female smoking. No significant difference was noted in the clinical outcome following embryo transfer. A study of larger sample size is required to evaluate whether the effects of cigarette smoking are independent of socioeconomic status and other related factors and whether they result in reduced ongoing clinical pregnancy and live birth rates.     

Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality

BACKGROUND: The purpose of the present study was to investigate the morphology of in-vitro matured metaphase II (MII) oocytes and to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of in-vitro matured MII oocytes was related to their subsequent fertilization and cleavage rates and to embryo quality. METHODS: This retrospective study included 264 MII oocytes obtained in 100 consecutive cycles. Oocyte retrieval was performed transvaginally and cumulus enclosed oocytes were matured for 28--30 h before evaluation. Prior to ICSI, all MII oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly (n = 144, 54%), grade II: oocytes with one anomaly (n = 87, 33%) and grade III: oocytes with at least two anomalies (n = 33, 12.5%). RESULTS: Oocyte grades did not differ between women with polycystic ovaries [grades I, II and III respectively: 58/94 (61.7%), 29/94 (30.9%) and 7/94 (7.4%)] and women with normal ovaries [grades I, II and III respectively: 86/170 (50.6%); 58/170 (34%); 26/170 (15.3%)]. Morphology was not related to fertilization rates. The cleavage rate was, however, affected by morphological anomalies (grade I [77/144 (53.5%) versus grade II 33/87 (37.9%) (P = 0.03)], although no significant decrease in cleavage rate could be demonstrated when all grade II and III oocytes were compared with normal oocytes. Significantly more embryos of good quality developed after grade I oocytes [54/144 (37.5%)] compared with those from grade II and grade III oocytes (22/120; P = 0.001). The presence of cytoplasmic abnormalities significantly decreased the cleavage rate (P = 0.04) and also the number of good quality embryos (P < 0.001). CONCLUSION: The in-vitro maturation of oocytes without anomalies yields higher quality embryos, with higher cleavage rates, than those with anomalies.     

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: 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.     

Improved methods for preparation of culture media for in-vitro fertilization and gamete intra-Fallopian transfer

A new method for preparation of culture media for IVF-ET andGIFT was developed which eliminated the requirement for volumetricsand glassware. Water weight was used instead of volumetricsfor preparation of media. Media prepared by the volumetric andwater weight methods were compared for (i) preparation time,(ii) pH, (iu) osmolarity and (iv) the percentage of two-cellmurine embryos developing to blastocysts. The time requiredfor preparation of media was significantly less for the waterweight method. Following equilibration with 5% CO², no differenceswere observed between the two methods for pH, osmolarity ordevelopment of embryos to the blastocyst stage. Time for mediapreparation and osmolarity was less variable among preparationdays for the water weight method. These results suggest thatmedia can be prepared more efficiently and precisely with thewater weight method than with the standard volumetric methodused by most IVF laboratories. The former method eliminatesconsiderable technician time which must be devoted to propercleaning/sterilization of volumetrics and the possibility ofmedia contamination by residual substances remaining on volumetricsfollowing improper cleaning.     

Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer.

The effect of macromolecules on mouse embryo development and viability after culture in sequential media was investigated. It was found that high rates of viable blastocysts could be obtained in the absence of any macromolecule. Blastocyst cell numbers were increased when bovine serum albumin was present in the culture medium, although this benefit was not manifest after blastocyst transfer. Rather, the highest rates of implantation and fetal development after blastocyst transfer were observed when hyaluronan was the macromolecule in the culture media. Subsequent analysis revealed that the beneficial effects of hyaluronan were due to its presence in the transfer medium. As the highest cell numbers and hatching rates obtained in this study occurred when both serum albumin and hyaluronan were present in the same medium, it is proposed that embryo culture media should contain both serum albumin and hyaluronan, while the transfer medium need only contain hyaluronan.     

The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts

Blastocysts from 198 patients were frozen using glycerol ascryoprotectant. No difference in the post-thaw survival of blastocystsor implantation rates was found between 177 patients (122 transfers)with all surplus embryos cultured to blastocysts before freezingand 20 patients (12 transfers) whose embryos were consideredunsuitable for freezing during cleavage and were then frozenas blastocysts. Nineteen pregnancies were achieved, of whichsix aborted. Pre-freezing morphology was similar in blastocystsof patients in groups 1 and 2 and did not relate to their survivalafter cryopreservation. A significantly lower proportion withsuspected damage after thawing was present among patients becomingpregnant after transfers of single blastocysts (P < 0.01)and implanting embryos were in general more expanded at thetime of transfer. No differences were detected between blastocystsresulting in normal development and those leading to abortion.The developmental consequences of damage to human blastocystsare discussed.