Human Tubal Fluid Medium Research Articles

Confirmation of the Beneficial Effects of Brief Coincubation of Gametes in Human In Vitro Fertilization

Objective: To confirm whether brief exposure of human oocytes to spermatozoa in vitro results in equivalent fertilization rates and possibly better quality embryos than overnight coincubation and to determine if there was a difference in outcome with regard to the type of culture medium used. Design: Prospective distribution of gametes between treatments in sequential patients. Setting: Assisted reproductive technology program in private hospital. Patient(s): Consecutively treated subfertile couples entering an infertility program. Intervention(s): Assisted reproductive technology treatment for infertility involving oocyte retrieval and in vitro fertilization. Main Outcome Measure(s): When possible, the outcome of fertilization and embryo quality were compared when gametes were coincubated for 1 hour or overnight. Two different formulations of human tubal fluid were compared in some cases. Result(s): There was no statistically significant difference in fertilization rates between a brief or overnight coincubation of gametes or between the two treatment groups with regard to the type of culture medium used. The quality of the embryos was significantly better in the 1-hour exposure group. The embryos in Basal XI human tubal fluid medium were of significantly better morphological quality than their siblings in D3+ human tubal fluid medium. Conclusion(s): Coincubation of oocytes and spermatozoa for a shorter period produced embryos of superior morphological quality than the generally accepted overnight protocol. A simple glucose and phosphate-free human tubal fluid medium resulted in early cleavage embryos of better morphological quality than a medium supplemented with glucose, taurine, and glutathione.

Ofloxacin: the next generation of antibiotic in sperm and embryo cultures for assisted reproductive technologies

Objective: To analyze the effect of different concentrations of ofloxacin on sperm kinematic parameters and to determine the embryotoxicity of ofloxacin at physiologic and at 100× concentrations. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Pooled cryopreserved donor sperm ( n = 7). Intervention(s): Human sperm were processed through two-layer discontinuous Percoll gradients, and the resultant pellet was resuspended in either HEPES-buffered human tubal fluid medium containing different concentrations of ofloxacin or the control medium. After measuring the kinematic parameters, the percentages of apoptosis and viability were obtained. Next, the sperm DNA was extracted and polymerase chain reaction of P-globin gene was performed followed by denaturing gradient gel electrophoresis. Mouse embryos recovered at the one-cell pronuclear or zygote stages were cultured in the presence or absence of ofloxacin up to the hatched blastocyst stage and differences in development were recorded. Main Outcome Measure(s): Sperm kinematic parameters, sperm P-globin gene, and number of embryos reaching the hatched blastocyst stage. Result(s): The number of embryos exposed to control and physiologic ofloxacin concentrations showed comparable excellent growth. However, the 100× concentration significantly arrested development. Rates of sperm viability and apoptosis measured 48 hours after exposure to the above concentrations were not different from controls. No differences were noted in the sperm kinematic parameters of sperm exposed to ofloxacin concentrations (1 X, 10×, and 100X) or control medium after 0, 4, and 48 hours of incubation. Denaturing gradient gel electrophoresis of β-globin genes from DNA exposed to varying ofloxacin concentrations failed to show any point mutations. Conclusion(s): Ofloxacin was embryotoxic at pharmacologic concentrations (100×). At physiologic or higher concentrations, ofloxacin appears to be safe and does not affect sperm kinematic parameters when compared with controls. This may indicate that sperm motility parameters alone cannot be relied on to evaluate the effects of drugs on fertility and that in vitro embryologic studies are essential. Ofloxacin at any concentration did not alter the rates of sperm apoptosis or viability. Ofloxacin does not appear to be mutagenic as evidenced by the β-globin gene analysis.

Cumulus cells are required for the increased apoptotic potential in oocytes of aged mice.

Recent studies with female ICR mice have suggested that oocyte DNA fragmentation is one reason for poor oocyte quality and lower fertility associated with ageing. Since it was not determined if this increased 'apoptotic' potential in aged oocytes is due to changes within the oocyte itself or within the microenvironment of cumulus cells (CC) surrounding the germ cell, we sought to clarify if CC were required to affect the rate of apoptosis in oocytes maintained in vitro. Intact cumulus-oocyte complexes (COC) were retrieved by superovulation of virgin female ICR mice at 7 weeks ('young') or 34-35 weeks ('aged') of age. One-half of the COC in each group were incubated at 37 degrees C in human tubal fluid medium under paraffin oil for 24 h. The other half of the COC in each group were denuded of CC and incubated under the same conditions (denuded oocytes; DO). Following incubation, COC were stripped of adherent CC by gentle pipetting. All DO were then fixed and checked by light microscopy for morphological changes characteristic of apoptosis. In young mice, the presence of CC had no significant effect on oocyte death rate (18 +/- 9% and 14 +/- 6% apoptotic oocytes in COC and DO, respectively; P > 0.05). However, in aged mice the percentage of CC-enclosed oocytes that underwent apoptosis was significantly greater as compared to the death rate in DO (48 +/- 3% versus 19 +/- 8% apoptotic oocytes, respectively; P < 0.05). This increased death potential was due to the presence of CC since the occurrence of apoptosis in DO of aged versus young mice was not significantly different (19 +/- 8% versus 14 +/- 6% apoptotic oocytes, respectively; P > 0.05). These results demonstrate that the age-dependent acceleration of apoptosis in oocytes maintained in vitro requires the CC.

Effect of different concentrations of amino acids in human serum and follicular fluid on the development of one-cell mouse embryos in vitro.

As a model for establishing an optimized medium for human in vitro fertilization (IVF), modified human tubal fluid (HTF) media containing amino acids at concentrations found in human serum and follicular fluid were prepared, and the effect of the media on development of random-bred (ICR) and F1 hybrid (CBF1) mice embryos was studied. The total concentrations of amino acids found in serum and follicular fluid were about one-third to one-half the concentrations present in two conventional media used in human IVF: Ham's F-10 and Eagle's minimal essential medium (MEM). When ICR mouse embryos were cultured in the HTF medium containing 21 amino acids at concentrations found in follicular fluid, the number of embryos developing to morulae at 72 h and to blastocysts at 96 h increased in comparison with those cultured in HTF medium. When HTF containing amino acids at concentrations found in serum was used, only induced morula formation at 72 h was enhanced. The number of hatching blastocysts at 96 h also increased when CBF1 mouse embryos were cultured with HTF supplemented with amino acids at concentrations found in follicular fluid. When ICR mouse embryos were cultured in modified HTF media containing concentrations of amino acids found in Ham's F-10 and MEM that contained higher concentrations of glutamine, embryo development was inhibited. The amount of ammonium produced during incubation for 3 days was significantly less when embryos were cultured in media containing concentrations of amino acids found in follicular fluid compared with when Ham's F-10 or MEM was the culture medium. Ammonium is produced by the breakdown of glutamine in the culture medium during incubation with or without embryos. These results suggest that the concentrations of amino acids found in follicular fluid are more effective and safer for embryo culture than those in other media currently in use.

Positive effect of partial zona-pellucida dissection on the in vitro fertilizing capacity of cryopreserved C57BL/6J transgenic mouse spermatozoa of low motility.

Although cryopreservation of mouse spermatozoa has recently become available for use, as yet there are considerable differences in fertilization efficiency of cryopreserved spermatozoa among various mouse strains. In this study, oocytes subjected to partial dissection of the zona pellucida (PZD) were inseminated with frozen-thawed C57BL/6J mouse spermatozoa. At 30, 60, 120, and 240 min after insemination, the oocytes were washed in human tubal fluid medium and cultured for 3-6 h. The fertilization rates of the PZD oocytes in each group at 6-7 h after insemination were significantly higher than that of the zona-intact control (73-88% vs 12%, respectively) (p < 0.01); but the incidence of polyspermy was nevertheless quite low (1.3-2.4%). The development rates of the monospermic oocytes to the morula and early blastocyst stages were in the 87-92% range, with 31-40% of those developing into offspring after embryo transfer. When the cryopreserved spermatozoa of C57BL/6J transgenic mice were used to fertilize PZD oocytes, the fertilization rates were as high as (73-76%) those of the PZD oocytes inseminated with the cryopreserved C57BL/6J spermatozoa, with 30-31% of the morulae and early blastocysts derived from the monospermatic oocytes developing into offspring. These results indicate that PZD of oocytes may provide an alternative when the fertilizing capacity of mouse spermatozoa has been compromised by cryopreservation.

The supplementation of culture medium with protease improves the hatching rate of mouse embryos.

Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition. These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus. In order to evaluate the effects of proteases on embryonic development and hatching, 2-cell mouse embryos were cultured for 72 h with or without proteases. The addition of 1.0 microg/ml pronase (PE) and/or 0.1 microg/ml proteinase K (PK) did not affect embryonic development up to the blastocyst stage (94.1% versus 88.2%; 92.2% versus 90.2%, respectively) but significantly increased the hatching rate (60.4% versus 39.2%, 71.8% versus 35.3%, respectively). However, the addition of alpha-chymotrypsin (Chymo) was detrimental to embryonic development and hatching. Changes in the structure of the zona pellucida (ZP) structure of embryos which had been cultured in human tubal fluid (HTF) medium with PE and PK were assessed by fluorescein isothiocyanate-conjugated (FITC)-casein. Embryos cultured in HTF-PE and PK were not stained with FITC-casein. When these embryos were cultured within oviducts, their perivitelline space (PVS) became strongly stained with FITC-casein which was easily removed by phosphate-buffered saline washing. This suggests that PE and PK altered the structure of the ZP. We suggest that the addition of PE and PK to culture media may accelerate the hatching of embryo, by structurally altering the ZP and PVS. This may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

Sperm interaction with fallopian tube apical membrane enhances sperm motility and delays capacitation

Objective: To understand the effect of sperm contact with the apical plasma membrane of tubal epithelial cells on sperm motility, velocity, and capacitation. Design: Prospective, controlled in vitro study. Setting: University medical center. Patient(s): Women of reproductive age undergoing hysterectomy for benign gynecologic indications and normozoospermic donors of proved fertility. Main Outcome Measure(s): Sperm motility as measured manually, velocity as measured by computer-assisted sperm motility analysis, and capacitation status as measured by the chlortetracycline fluorescence assay. Result(s): Sperm incubated with apical membrane vesicles had a significantly higher motility at 12 (87.4% ± 3.4% versus 69.2% ± 4.8% [mean ± SEMI), 24 (85.2% ± 3.1% versus 60.5% ± 7.2%) and 48 hours (78.9% ± 5.3% versus 42.4% ± 11.3%) compared with control (sperm incubated with human tubal fluid media in the absence of apical membrane vesicles) (n = 4). Progressive velocity was significantly higher at 12 (78.2 ± 1.4 versus 61.7 ± 16.1μm/s) and 24 (66.2 ± 3.9) versus 34.4 ± 9.8μm/s) hours (n = 4). Incubation with apical membrane vesicles significantly slowed the transition from uncapacitated to capacitated chlortetracycline fluorescence pattern (n = 5). Conclusion(s): Contact with the apical plasma membrane of tubal epithelial cells enhances sperm motility and delays sperm capacitation in vitro.

P-089. Comparison of a commercially available glucose/phosphate-free medium and human tubal fluid medium for IVF

P-089. Comparison of a commercially available glucose/phosphate-free medium and human tubal fluid medium for IVF

Antibiotics: effect on cryopreserved-thawed human sperm motility in vitro

Objective: To analyze the motility and fertilizing capacity of sperm treated with different antibiotics. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Pooled cryopreserved donor sperm (n = 14). Intervention(s): Sperm were washed with Percoll and resuspended in HEPES-buffered human tubal fluid medium containing either amoxicillin, of ioxacin, ciprofloxacin hydrochloride, nitrofurantoin monohydrate, doxycycline hyclate, cefuroxime axetil, or control medium. Main Outcome Measure(s): Sperm kinematic and fertilizing parameters. Result(s): Sperm hyperactivation was decreased in physiologic concentrations of ciprofloxacin hydrochloride and doxycycline hyclate over the course of 48 hours. At pharmacologic concentrations, ciprofloxacin hydrochloride, cefuroxime axetil, and nitrofurantoin monohydrate adversely affected motility with decreased rapid progression. Cessation of motility occurred in cefuroxime axetil and nitrofurantoin monohydrate. Sperm hyperactivation was also absent. Cefuroxime axetil decreased the percentage of intact acrosomes. In contrast, physiologic doses of ciprofloxacin hydrochloride or of ioxacin enhanced sperm fertilizing capacity. Conclusion(s): Ciprofloxacin affected hyperactivation by altering membrane properties, whereas doxycycline inhibited the capacitation process. Cessation of motility in cefuroxime axetil was linked to disrupted sperm head membranes. Sperm motility and fertilizing capacity were decreased in nitrofurantoin because of decreased metabolism. The positive effect of of ioxacin on fertilizing capacity did not involve changes in acrosome.

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

Culture with Matrigel inhibits development of mouse zygotes.

It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. Matrigel may be a deleterious addition to embryo culture or coculture systems.

Birth of a western lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer.

A 21-year-old multiparous female exhibiting 31-41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocytes (n = 11) were placed in modified human tubal fluid (mHTF, 100 microliters) medium under oil at 37 degrees C in humidified 5% CO2. Frozen semen, collected by voluntary ejaculation, was thawed (70 degrees C H2O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247-260, 1997.

Globulin‐Enriched Protein Supplements Shorten the Pre‐Compaction Mitotic Interval and Promote Hatching of Murine Embryos

To determine whether Synthetic Serum Substitute (SSS), which contains human globulins in addition to Human Serum Albumin (HSA), is superior to HSA alone as a protein supplement for embryo culture. Development of mouse zygotes to eight-cell/compacting morulae and to hatching/hatched blastocysts was assessed in Human Tubal Fluid (HTF) medium containing either SSS of HSA. Although there was no difference in the overall blastocyst rate at 120 h in HTF+SSS versus HTF+HSA, significantly more embryos at 54 h were at the eight-cell/compacting morula stage in HTF+SSS. At 120 h, there were more hatching/hatched blastocysts in HTF+SSS, and hatching correlated with SSS concentration. Addition of isolated globulins to HSA significantly stimulated the number of hatching/hatched blastocysts. Hatching could be "rescued" by transfer of embryos grown in HTF+HSA to globulin-containing media and prevented by removal of globulins as late as the compacted morula stage (54 h). SSS is superior to HSA alone for embryo culture. The stimulatory effects on mitosis and hatching may be mediated directly by globulins or by other components in the globulin-enriched fraction.

Insulin-like growth factor-binding proteins produced by Vero cells, human oviductal cells and human endometrial cells, and the role of insulin-like growth factor-binding protein-3 in mouse embryo co-culture systems.

Co-culturing embryos on helper cells can mimic the in-vivo environment, thereby enhancing embryo development in vitro. Insulin-like growth factors (IGF) and their binding proteins (IGFBP) also enhance embryo development. To investigate the kinds of IGFBP produced by various cell monolayers and the effects of IGFBP-3 on mouse embryo co-culture systems, 2-cell ICR mouse embryos were cultured in either human tubal fluid medium alone or in the presence of Vero cells, human oviductal cells or endometrial cells. The helper cells were analysed immunohistochemically to investigate the types of IGFBP produced by various cell monolayers. The concentrations of IGF-I and IGFBP-3 in media obtained from the culture of embryos alone, cells alone or cells plus embryos were determined by radioimmunoassays. On day 7, more blastocysts hatched in the co-culture groups (73% in the Vero cell group, 76% in the endometrial cell group and 74% in the oviductal cell group) than in the control group (43%) (P < 0.0001). The results of immunohistochemistry revealed that (i) all three cell groups produced a lot of IGFBP-1, -2 and -3, but only a little of IGFBP-4 and -5; and (ii) IGFBP-1, -2, and -3 were present in blastocysts in either the presence or absence of helper cells. The IGF-I secreted by cell monolayers or embryos was undetectable (detection limit 0.83 microg/l). The IGFBP-3 concentrations in media obtained from co-cultured embryos and cells were significantly higher than in media without embryos (median values in oviductal cell culture medium, 165 versus 127 microg/l, P = 0.04; median values in endometrial cell culture medium, 277.5 versus 183.5 microg/1, P = 0. 0002; median values in Vero cell culture medium, 219 versus 120 microg/l, P = 0.011). Although IGFBP-3 concentration in the medium that contained embryos alone was undetectable by radioimmunoassay (detection limit 1.1 microg/l), immunohistochemistry demonstrated the presence of IGFBP-3 in the embryos. Co-culture in systems in which there was an increased production of IGFBP-3 led to an improved development of mouse embryos. IGFBP can improve the binding of IGF to cell surface receptors of target tissue, and thus enhance the effect of limited IGF concentrations in promoting embryo development in a co-culture system. We conclude that Vero cells, human endometrial cells and oviductal cells produce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a role in embryotrophic potential by either regulating the action of IGF or directly enhancing embryo development.

In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium.

We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa.

Influence of elevated pH levels on structural and functional characteristics of the human zona pellucida: functional morphological aspects.

A total of 86 fresh and salt-stored immature human oocytes derived from postmortem ovarian tissue were used for this study. Oocytes were randomly incubated either in synthetic human tubal fluid medium (untreated zonae) or in a chemically defined medium (treated zonae). Sperm binding experiments using hemizona assay conditions exhibited a 10-fold increased binding of sperm to treated compared to untreated oocytes (272.7 +/- 43 versus 24.3 +/- 15 sperm bound, respectively; P < 0.0001). pH recordings during incubation showed elevated pH levels of 8.1 compared to pH 7.2 among treated and untreated zonae, respectively. Ultrastructural examination showed a spongy appearance of the surface of treated zonae, whereas untreated zonae appeared compact with smooth surface. The marked increase in sperm binding among treated zonae, together with the ultrastructural findings, suggest that the altered zona surface enhances sperm binding. The physiological maturational process of the zona pellucida might be manipulated in vitro, thus increasing sperm binding to the zona.

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-culture of unfertilized (degenerating) oocytes on the development of in-vitro fertilized (IVF) mouse embryos. In experiment 1, groups of one, five, 10 or 20 zygotes were cultured in 20 microliter drops of modified human tubal fluid (HTF) medium for 168 h at 38.7 degrees C in 5% CO2 and 95% air. As the embryo density increased, significantly (P < 0.05) higher rates of embryos reached hatched blastocyst stage. In addition, the time required for hatching after IVF was significantly (P < 0.05) shortened by the increase in embryo density. In experiment 2, 10 IVF zygotes were cultured with or without 10 unfertilized (degenerating) oocytes in 20 microliter drops of HTF medium. The rates of IVF embryos that developed to morula, blastocyst, expanded blastocyst and hatched blastocyst stages were decreased significantly (P < 0.01) by culturing embryos with unfertilized oocytes compared with culturing embryos alone. In experiment 3, groups of one or 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocytes were cultured in 20 microliter drops of HTF medium and the number of cells per blastocyst was examined at 120 h after IVF. Increasing embryo density resulted in a significant (P < 0.05) increase in the number of cells per blastocyst. In contrast, the cell number of IVF embryos that developed to blastocyst decreased significantly (P < 0.05) when they were cultured with unfertilized oocytes. The results suggest that in-vitro development of IVF mouse embryos is enhanced by increasing embryo density and is impaired by co-culture with unfertilized (degenerating) oocytes.

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

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). 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 alpha- and beta-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). 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. Synthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.

Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions

To determine the effect of medium with or without glucose and phosphate on the fertilization and development of human oocytes. Sequential allocation of alternate patients to one of two treatment groups. Private practice infertility programs. Ten couples requesting treatment for infertility. Gametes from each couple were collected, washed, and incubated in one of two culture media under investigation. Number of oocytes collected, fertilized, cleaving, replaced, and implanting in each patient. Development of any supernumerary embryos to fully expanded blastocysts in vitro. There was a significant increase in the proportion of transferred embryos implanting in the group of patients whose gametes were handled in medium devoid of glucose and phosphate. All other comparisons of factors that may have influenced implantation rates between the two groups of patients were not significantly different. High rates of fertilization, cleavage, implantation, and development of supernumerary human embryos to the blastocyst stage in vitro were obtained with a modified human tubal fluid medium containing ethylenediaminetetraacetic acid and glutamine but devoid of glucose and phosphate ions. A prospective randomized trial is necessary to evaluate the clinical significance of these observations.

Fertilization and early embryolgoy: Can Matrigel substitute for Vero cells in promoting the in-vitro development of mouse embryos?

The influences of Vero cells and the basement membrane substratum for these cells (Matrigel) on the rate of hatched blastocyst formation from mouse zygotes in vitro were compared. Zygotes obtained from C57BL/6 x BALB/c F1 females pretreated with pregnant mare's serum gonadotrophin/human chorionic gonadotrophin mated with BDF1 males were cultured (120 h) in human tubal fluid medium supplemented 0.5% with bovine serum albumin. The rates of early hatching and hatched blastocyst formation at 96 and 120 h of culture were expressed as the percentage of 2-cell embryos visualized after the initial 24 h. The rate of total blastocyst formation did not differ between treatment groups. However, < 10% of embryos cultured for 96 h in medium alone advanced to the hatching stage compared with 35-40% of blastocysts cultured with Vero cells or with Matrigel alone. Similarly, by 120 h of culture, only 20% of embryos cultured in medium alone developed to hatching or hatched blastocysts compared with > 70% for those embryos co-cultured with Vero cells or with Matrigel. In conclusion, Vero cells improved the rate of development of mouse embryos to hatched blastocysts during serum-free culture. Similar improvements were seen in the presence of Matrigel alone; Matrigel is the basement membrane substratum used for the Vero cells. Further studies on the means whereby Matrigel promotes early embryonic development (e.g. appropriate combination of basement membrane-associated growth factors) may lead to a safe, defined medium preparation for the stimulation of in-vitro development of human embryos.