Alpha-minimum essential medium (MEM) enhances in vitro hatched blastocyst development and cell number per embryo over Ham's F-10

Problem The development of mouse embryos in vitro is affected primarily by mouse strain-genotype and culture conditions. Embryo culture studies evaluate the effectiveness of culture conditions in supporting one- or two-cell mouse embryo development to the blastocyst stage by reporting the percentage blastocyst formation rate.Method Determining the cell number per cultured blastocyst may also help in determining embryo culture medium quality. The objective of this study was to determine the effect of MEM and Ham's F-10 on overall CFW mouse embryo development and hatched blastocyst cell number in vitro. CFW embryos cultured in MEM had significantly higher (87%; P<0.001 hatched blastocyst rates than embryos cultured in F-10 (56%).Results A significant difference in nuclei per hatched blastocyst was found between MEM and F-10 (P<0.001). The results demonstrate that inbred mouse embryos have significantly higher blastocyst hatching rates and higher cell numbers per blastocyst when cultured in MEM.     

Minimum essential medium alpha (MEM) enhances assisted reproductive technology results. I. mouse embryo study

Purpose Our purpose was to find a medium to enhance mouse zygote development and, hopefully, to apply the results to a coculture system and to enhance the ART pregnancy rate.Design The study was designed to compare different media's support of mouse zygote development with/without serum supplement. The outcome measure was the percentage of mouse zygotes/embryos that developed to the expanded blastocyst and hatchout stage.Results (1) Using human tubal fluid (HTF), one-cell zygotes had a 34.6±5.2% (mean±standard deviation) development rate, and two-cell embryos a 86.5±3.2% development rate. (2) Minimum essential medium alpha (MEM) showed the best results (52.2±14.5%) among Ham's F-10 (19.1±6.3%), HTF (26.8±8.2%), NCTC-135 (38.8±12.6%), MEM with nuclei acid (24.6±10.0%), and Dulbecco's modified Eagle medium (28.0±20.2%). (3) With the serum supplement, there was no significant difference among Ham's F-10 (21.5±23.7), HTF (29.3±10.4%), NCTC-135 (36.5±6.2%), and MEM (38.8±17.9%).Conclusion MEM is the best medium among the six media examined. Preliminary study showed that MEM gave a good clinical pregnancy rate (29%).Some of the data presented at the 7th World Congress on In Vitro Fertilization and Assisted Procreation, Paris, France, June 30 to July 3, 1991.     

Peptides extracted from vero cell cultures overcome the blastocyst block of mouse embryos in a serum-free medium

Purpose The aim of this work was to evaluate the effect of a Vero cell coculture system on the development of mouse embryos. Methods Mouse embryos were randomly divided and cultured in human tubal fluid (HTF) medium with/without Vero cell monolayers, conditioned medium (CM) obtained from Vero cell cultures, and HTF medium supplemented with peptides extracted from CM. The concentrated CM was examined by SDS/PAGE. Results The development of mouse embryos was blocked at the blastocyst stage in pure HTF medium (1.4% hatching at day 5). This “blastocyst block≓ was overcome by coculture with Vero cell monolayers (48.1% hatching at day 5; 1.4 vs 48.1%; P<0.001). CM and the addition of 5% fetal bovine serum (24.1 and 34.9% hatching, respectively, at day 5) were also able to enhance the process of hatching. In the other experiment, the addition of peptides extracted from Vero cell cultures also overcame the blastocyst block (12.5%) compared with pure HTF medium (2.1%) (P<0.05). Electrophoretic separation revealed several classes of polypeptides consistently secreted into CM obtained from Vero cell cultures. Most peptides occurred in the M_r range between 6.5 kd and 35.9 kd. Conclusion A developmental block (blastocyst block) of mouse embryos in a serum- and protein-free medium (HTF) was discovered in this study. This block was effectively overcome by HTF plus serum and coculture with Vero cell monolayers and also by the peptides extracted from Vero cell-conditioned medium. We speculate that certain factors secreted or converted by Vero cells may be critical in hatching of mouse embryos. Further study of these factors may be helpful in delineating its mechanism.     

Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos

Purpose: We reported previously on the use of coculture with cumulus cells in insemination medium for the development of human embryos in vitro. Here we describe a prospective trial to determine if this procedure has a significant beneficial effect. Methods: On the day after insemination, zygotes were randomized for culture in either a fresh drop of medium without (– cum) or were left in their insemination drop with (+ cum) cumulus cells. Embryos with the best morphological quality were replaced on the third day of development at the eight-cell stage. The remaining embryos were cultured for a further 3 days and cryopreserved if they reached the fully expanded blastocyst (FEB) stage. Three different culture media were used over the period of this study. Results: In 11 patients, supernumerary embryos were available only for continued culture in + cum and three patients had embryos cultured in only – cum. Thirty-nine other patients had embryos assigned to both + cum and – cum treatments. In the + cum group, 98 blastocysts developed from 216 embryos cultured for 6 days (45%), and this was significantly greater (P<0.01) than the 48 blastocysts from 156 embryos (31%) developing in the absence of cumulus cells. In basal HTF medium (HTF medium with EDTA and glutamine) and basal XI HTF medium (similar to basal HTF but devoid of glucose and phosphate), culture of embryos with cumulus cells produced significantly more FEBs than in the absence of cumulus cells. There was no significant difference between the two culture treatments when regular HTF medium was used. Preliminary results indicate that pronectin-coated dishes provide a good substratum for cumulus cell attachment and embryo development. Conclusions: The culture of human embryos with their cumulus cells in insemination drops of medium produces a significantly greater proportion of FEBs than when the zygotes are transferred to fresh culture drops devoid of cumulus cells. This is the first report of a significantly higher blastocyst rate with coculture in which a real comparison has been made between two culture treatments which differ only in the presence or absence of homologous cumulus cells in insemination drops.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction. Vienna, Austria. April 3–7, 1995.     

The surplus human embryo: its potential for growth, blastulation, hatching, and human chorionic gonadotropin production in culture

Early embryos that were unsuitable for transfer to patients or for cryopreservation were cultured either in a human tubal fluid (HTF) or a minimum essential medium (MEM). A significantly higher proportion of embryos developed to blastocysts in MEM (26.8%) than in HTF (14.5%). Approximately similar proportions of embryos formed blastocysts in MEM in the presence or absence of serum. The rate of embryo growth to blastocysts was similar in all media. Blastocyst hatching occurred in MEM + or - serum, but it failed to occur in HTF with serum. Released human chorionic gonadotropin (hCG) from hatched and intrazonal blastocysts was detected by day 8 after fertilization. The mean amount of hCG produced by day 14 was 19,500 mIU from hatched and 1,550 mIU from intrazonal blastocysts. Serum stimulated the output of hCG.     

The use of Matrigel* at low concentration enhances in vitro blastocyst formation and hatching in a mouse embryo model

Objective: To determine the effect of Matrigel at a low concentration on the growth of mouse embryos in culture.

Design: Randomized case-control study of mouse embryos.

Setting: An academic research environment.

Animals: Mouse embryos.

Intervention(s): Embryos were cultured in Quinn’s or Celbio’s human tubal fluid (HTF) enriched with 1.5% bovine serum albumin and 0.8% liquid Matrigel. Each HTF was compared with the same medium devoid of Matrigel. Afterward, Quinn’s and Celbio’s HTF, both containing Matrigel, were compared directly. Embryos were cultured in four-well dishes, and their morphology and viability were assessed at 96 hours.

Main Outcome Measure(s): Level of interleukin-1 in media collected at the end of culture.

Result(s): In both types of HTF, the presence of Matrigel allowed a larger number of embryos to reach the blastocyst stage and to hatch; blastocyst morphology also was improved. These positive effects were enhanced in Quinn’s HTF: embryos cultured in its Matrigel-enriched version secreted a higher level of interleukin-1 than those in Celbio’s HTF plus Matrigel and also showed a better morphology.

Conclusion(s): In the mouse embryo model, Matrigel improves culture conditions in terms of both embryo viability and morphology, and these effects are enhanced in Quinn’s HTF.     

Anti-platelet activating factor (PAF) antibody inhibits CFW mouse preimplantation embryo development

Objective Our purpose was to investigate the effect of anti-PAF antibodies on CFW mouse embryo development in vitro.Design We studied the in vitro development of CFW mouse one-cell-stage embryos cultured in MEM supplemented with anti-PAF, anti-IgG, or MEM alone to the hatched blastocyst stage.Results Mouse embryos cultured with anti-PAF (15 dilution; 61%) significantly decreased embryo development compared to controls (MEM alone; 93%), whereas embryos cultured in anti-mouse IgG-supplemented MEM (1 10 dilution; 93%) had no effect.Conclusions The results provide additional evidence that PAF is produced and secreted by cleavage-stage embryos and is required during the preimplantation period.     

Synthetic serum substitute (SSS): A globulin-enriched protein supplement for human embryo culture

Objective The purpose of the present study was to evaluate whether an IVF protein supplement prepared from human serum albumin (HSA) and human globulins would retain performance characteristics equivalent to those reported for the commercial plasma expanders, Plasmatein (Alpha Therapeutics, Los Angeles, California) and Plasmanate (Cutter Biological, Miles Inc., Elkhart, Indiana).Methods Pronuclear-stage human embryos were randomly divided and cultured in human tubal fluid medium (HTF) supplemented with either HSA (5 mg/mL) or Plasmatein (10%, v/v; 5 mg/ml as a means of indirectly assessing the effect - and - globulins have on embryonic development. Those results coupled with the known composition characteristics of Plasmatein were used as the starting basis to formulate test lots of synthetic serum substitute (SSS).Results Significantly (P<0.05) more of the human embryos cultured in Plasmatein supplemented medium reached the four-cell or greater stage by 40 hr postinsemination than a comparable group cultured in HSA alone. Lot 1 of SSS, formulated with HSA (84% of total protein) and human globulins (16% of total protein) and an aqueous lipoprotein fraction derived from human plasma (Excyte IV; Miles Diagnostics, Kankakee, Illinois), produced accelerated early embryonic growth relative to control murine embryos grown in the presence of Plasmatein, however, the percentage of the embryos reaching the hatched blastocyst stage was decreased (45 vs 100%). Human embryos from seven patients, randomized to HTF medium supplemented with Plasmatein or lot 1 of SSS, showed equivalent growth at 36– 40 hr postinsemination. A microprecipitate developed in media supplemented with lot 1 after several days of culture. The Excyte IV concentration was reduced and, ultimately, eliminated from the subsequent and final prototype lots of SSS. Murine embryos grown in the presence of lipoprotein free SSS showed significantly accelerated (P<0.01) growth at 17 hr postthaw compared to Plasmatein and all embryos progressed to hatching by 41 hr. Human embryos, randomized to either Plasmatein or lot 3 of SSS, showed significantly accelerated growth (P<0.01) when scored at 38 hr following insemination.Conclusion Synthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.Irvine Scientific, Santa Ana, California.Presented in part at the Conjoint Annual Meeting of the American Fertility Society and Canadian Fertility and Andrology Society, Montreal Convention Center, Montreal, Quebec, Canada, October 9–14, 1993.     

Clinical Assisted Reproduction: Prolonged Culture of Human Cryopreserved Embryos with Recombinant Human Leukemia Inhibitory Factor

Purpose: To evaluate the efficiency of recombinant humanleukemia inhibitory factor (LIF) in the prolonged culture ofhuman cryopreserved-thawing embryos. Methods: After thawing, all embryos were divided into fourgroups: (1) Human tubal fluid (HTF), (2) HTF + LIF, (3)M3^TH medium, and (4) M3^TH medium plus LIF. Followingprolonged culture, embryo development in each groupwas compared. Results: In embryo development from about the 2– to 4–cellto 9– to 16–cell stage, there were nonsignificant differencesbetween each group. There was lower morula formation ratein group 1 (6.9%) than those in other groups (23.2%, 19.7%,23.1%). The lower blastocyst formation in group 1 and 3(0%, 0%) than those in group 2 and 4 (11.0%, 12.8%)were noted. Conclusions: LIF is beneficial for preimplantation embryos.LIF does not influence the early embryo development.LIF-supplemented HTF provided a similar culture environmentfor thawing embryos as LIF-supplemented M3^TH medium.Supported by China Medical College Hospital, Taiwan     

Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin

The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts.     

Evaluation of synthetic serum substitute versus serum as protein supplementation for mouse and human embryo culture

Purpose: Our purpose was to determine the effect of Synthetic Serum Substitute (SSS) versus serum supplementation on fertilization rates and subsequent development of embryos from patients undergoing IVF.Procedure: Experiment I compared the effects of SSS to human serum on mouse embryo development. Two hundred one-cell B6D2F1 mouse embryos were cultured in 100-µl droplets of human tubal fluid (HTF) containing either (1) no protein (control;n=37), (2) 15% serum from women with tubal infertility (n=44), (3) 15% serum from women with endometriosis (n=49), (4) 15% fertile donor serum (n=33), or (5) 15% SSS (n=37). Experiment II compared the effects of SSS to human serum on the development of embryos from patients undergoing IVF. Thirty-three women were included in this study. A total of 371 oocytes was cultured in HTF containing either (1) maternal or donor serum (n=140) or (2) 15% SSS (n=231). Embryo development was evaluated 48 hr after fertilization.Results: In Experiment I, the rate of blastocyst development was evaluated at 48, 72, and 96 hr of culture. Sixty-four and nine-tenths percent of embryos cultured in SSS were morulae at 48 hr of culture (versus 5.4, 0, 8.2, and 6.1 in Groups 1, 2, 3, and 4, respectively). By 72 hr, 29.7% of these embryos had developed into blastocysts (versus 0, 0, 8.2, and 3.0, for Groups 1, 2, 3, and 4, respectively). This percentage increased to a total of 83.7 after 96 hr (versus 27.0, 20.4, 38.8, and 39.4 for Groups 1, 2, 3, and 4, respectively). Forty-three and two-tenths percent of the blastocysts cultured in SSS had hatched from their zonae by 96 hr. With the exception of Group 5, which had a rate of 9.1%, embryo hatching was not observed in any of the groups at the termination of culture (96 hr). In Experiment II there were no differences in cell stage or quality of human embryos cultured in SSS or serum, but fertilization rates tended to be better (P=0.07) for oocytes inseminated in media containing SSS (70.0%, vs 55.0% for serum).Conclusions: SSS appears to be a superior protein source for mouse embryo growth and is as good as serum from fertile donors in promoting in vitro human embryo development.     

Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system

Purpose A noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-sec pulses at a wave-length of 2.1 m and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two-to four-cell FVB mouse embryos.Results In the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO₂ humidified air incubator at 37°C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group.Conclusions These preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.     

Comparison of four media types during 3-day human IVF embryo culture

The aim of this study was to compare the effectiveness of human tubal fluid (HTF), G1.2, Sage Cleavage and Life Global media for IVF outcome during 3-day culture of human embryos. A three-phase auto-controlled study was conducted in which IVF outcome was compared between (1) HTF and G1.2, (2) HTF and Cleavage, and (3) Cleavage and Life Global. In phase 1, no differences in embryo quality were observed between HTF and G1.2. However, embryos derived from intracytoplasmic sperm injection (ICSI) displayed significantly improved quality when grown in HTF versus G1.2. No differences in pregnancy and implantation rates were observed in cases where embryos transferred were grown exclusively in HTF or G1.2 media. In phase 2, embryo quality was significantly improved for embryos cultured in Cleavage versus HTF media (P < 0.001). However, pregnancy, implantation and spontaneous abortion rates were similar between the two media. In phase 3, there were no differences in embryo quality, pregnancy, implantation, and spontaneous abortion rates between Cleavage and Life Global media. Overall, the data indicate that Life Global and Cleavage media yield similar results in a 3-day IVF culture programme. Cleavage medium is superior to HTF, as evidenced by significantly improved embryo quality (P < 0.001). Meanwhile, HTF medium is superior to G1.2 for ICSI cases.     

Effects of transfer of embryos independently cultured in essential and sequential culture media on pregnancy rates in assisted reproduction cycles

Purpose

Several culture media are available to be used in ART. However it is uncertain whether embryos would preferably benefit from one type of medium or the association of different media.

Methods

We performed this study to evaluate the impact of simultaneous transfer of embryos independently cultured in two distinct culture media, on pregnancy outcome. A total of 722 couples who underwent infertility treatment were sequentially allocated into three groups: those who had half of the embryos individually cultured in MEM and the other half cultured in sequential media (MEM + Seq Group) (n = 243); those who had all embryos cultured only in sequential medium (Seq Group) (n = 239); and those who had all embryos cultured only in MEM (MEM Group) (n = 240).

Results

The pregnancy rate was higher in the MEM + Seq group (51.8 %) than the Seq group (36.7 %) (p < 0.001). However the pregnancy rate observed in the MEM group was similar to the others (44.2 %). When a logistic regression test was applied it demonstrated that the number of transferred embryos did not interfere in the pregnancy rates.

Conclusions

Our results suggests that offering different culture conditions for sibling embryos with subsequent transfer of embryos that were kept in distinct culture media, might increase pregnancy rates in assisted reproduction cycles.     

Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated. Methods: Cumulus-oocyte-complexes (COCs) were matured in IVM medium with serum. Presumptive zygotes were cultured in serum free in vitro culture (IVC) medium. Embryos were used as chilled or control samples at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Embryos in 0.2 mL PBS in plastic straws were cooled rapidly in ethanol baths at 0°C for 30 min. Embryo viability was assessed by in vitro development. Results: The percentage of control embryos that hatched as blastocysts increased the later stage at which they were selected. Relative proportion of embryos increased from 28% to 48% to 68% when chilled at the 8-cell, morula or blastocyst stages. Conclusions: IVF-produced embryos are differentially susceptible to cooling injury. Cell counts made of those blastocysts formed from chilled embryos indicated subtle effects of chilling.     

Initial experience with extended culture and blastocyst transfer of cryopreserved embryos

Objective: Our purpose was to evaluate the viability and transfer efficiency of cryopreserved embryos allowed to develop into blastocysts in extended culture for in vitro fertilization. Study Design: The embryos for in vitro fertilization that had been cryopreserved at either 2 PN (pronuclear) or cleaving stage (day 1-3) were thawed and cultured for uterine transfer on day 5. Outcome for day 5 embryo transfer was prospectively compared with previous outcomes from embryos transferred on day 2 or 3. Results: For embryos thawed and transferred on day 2 or 3 (n = 99), the pregnancy rate was 33%, the implantation rate per embryo transferred was 15.2%, and the rate of multiple gestations was 42.4% (14/33) with 35.7% of pregnancies having ≥3 gestational sacs. For extended culture embryos transferred on day 5 (n = 25), the pregnancy rate was 36%, the implantation rate per embryo transferred was 16.7%, and the rate of multiple gestations was 33.3% (3/9) with all of these being twins. For embryo transfers performed on day 5 in which only blastocysts were transferred (n = 9), the pregnancy rate was 66.7%, the implantation rate per blastocyst was 44.4% (greater than the rate for the day 2 or 3 embryos, P = .0043), and the rate of multiple gestations was 33.3% (2/6) with all of these being twins. In extended culture 29.8% of cryopreserved embryos progressed to the blastocyst stage. In this series 4 subjects (15.4%) did not have blastocysts by day 5. Conclusion: Acceptable pregnancy rates can be obtained from cryopreserved embryos cultured to the blastocyst stage with a significantly higher implantation rate. Transfer of embryos that have “self-selected” to blastocysts results in reduced risk of higher-order (>2) multiple gestations, because only 1 or 2 embryos are transferred. (Am J Obstet Gynecol 1999;180:1472-4.)     

Laser micromanipulation in the mouse embryo: a novel approach to zona drilling.

OBJECTIVE: To introduce the use of excimer lasers for penetration of the zona pellucida for micromanipulation purposes. DESIGN: Cryopreserved two-cell mouse embryos were thawed and exposed to the 248-nm line of a krypton fluoride excimer laser (Lambda Physik EMG 202, Goettingen, Germany) creating a 2 to 4-micron opening in the zona pellucida. SETTING: The Laser Ablation Laboratory at DuPont and the in Vitro Fertilization Laboratory at The Medical Center. INTERVENTIONS: The embryos were exposed in either phosphate-buffered solution (PBS) or modified human tubal fluid (HTF) with the laser power varying from 1 to 2 J/cm2 and cultured in Ham's F-10 medium (GIBCO, Grand Island, NY) with 0.4% bovine serum albumin. MAIN OUTCOME MEASURES: The outcome of each experiment was measured by blastocyst formation of laser-exposed embryos as compared with a set of unexposed control embryos handled in a similar fashion. RESULTS: Successful laser penetration of the zona pellucida was achieved using the 248-nm line of a krypton fluoride excimer laser. A higher blastocyst formation was found for embryos exposed in PBS. The higher optical absorption of the modified HTF partially inhibited embryo development. The blastocyst statistics increased 2.5-fold times by reducing the exposure of the embryos to ablation by-products. CONCLUSIONS: The use of a krypton fluoride excimer laser was introduced as a new method to open the zona pellucida of two-cell mouse embryos without interrupting blastocyst formation.     

Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing

Purpose To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. Results In the first experiment, unfertilized oocytes that were frozen in 1.5 Mdimethylsulfoxide (DMSO) supplemented with 0.2 Msucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4‡C than at room temperature (39.4 vs 19.4%; P<0.01). The addition of EDTA (10 ΜM)to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P<0.01). Conclusion Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 MDMSO and 0.2 Msucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.     

Preimplantation murine embryos are more resistant than human embryos to bacterial endotoxins

Bacterial endotoxins have been correlated with increased fragmentation of early cleavage-stage human embryos and decreased pregnancy rates in human in vitro fertilization programs. The purpose of the present study was to test the direct effects of increasing concentrations of endotoxins on in vitro fertilization and development of mouse embryos to the blastocyst stage. Sexually mature B6D2F1 female mice were superovulated and oocytecumulus complexes were collected from the oviducts and randomized into control and treatment groups. Oocytes (n=867) were inseminated with capacitated sperm. Treatments included Ham's F-10 supplemented with 3 mg/ml bovine serum albumin and increasing amounts of endotoxin (0.35, 0.64, 0.92, 1.5, 2.08, 3.21, 6.07, and 11.79 ng/ml). Percentage cleavage, percentage fragmentation at the four-cell stage, and percentage expanded blastocyst formation (of cleaved embryos) were evaluated. Statistically significant decreases in cleavage at 6.07 ng/ml (P<0.05) and blastocyst formation at 11.79 ng/ml (P<0.05) of endotoxin were observed. Fragmentation at the four-cell stage was significantly increased at 3.21 ng/ml (P<0.05) of endotoxin. We conclude that the levels of endotoxin necessary to decrease murine preimplantation development significantly is higher than that reported for human embryos.     

Dexamethasone attenuates the embryotoxic effect of endometriotic peritoneal fluid in a murine model

Purpose

The in vitro fertilization (IVF) pregnancy rate of women with advanced stage endometriosis is nearly half that of the general population, suggesting incomplete targeting of the pathophysiology underlying endometriosis-associated infertility. Compelling evidence highlights inflammation as the etiologic link between endometriosis and infertility and a potential target for adjunctive treatment. The objective of this study was to examine the effect of dexamethasone on murine embryos exposed to human endometriotic peritoneal fluid (PF) using the established murine embryo assay model.

Methods

PF was obtained from women with and without severe endometriosis. Murine embryos were harvested and randomly allocated to five groups of culture media conditions: (1) human tubal fluid (HTF), (2) HTF and 10 % PF from women without endometriosis, (3) HTF and 10 % PF from women with endometriosis (PF-E), (4) HTF with PF-E and 0.01 mcg/mL dexamethasone, and (5) HTF with PF-E and 0.1 mcg/mL dexamethasone. Embryos were cultured in standard conditions and evaluated for blastocyst development.

Results

A total of 266 mouse embryos were cultured. Baseline blastulation rates were 63.6 %. The addition of peritoneal fluid from women with endometriosis decreased the blastocyst development rate to 38.9 % (P = 0.008). The addition of 0.1 mcg/mL of dexamethasone to the culture media restored the blastulation rate to near baseline levels (61.2 %; P = 0.019).

Conclusions

The results of our in vitro study demonstrate the capacity of dexamethasone to mitigate the deleterious impact of endometriotic PF on embryo development. If confirmed in vivo, dexamethasone may prove a useful adjunct for the treatment of endometriosis-associated infertility.