Fetal Cord Serum Research Articles

Effects of human serum supplementation on fertilization and development of mouse embryos.

Serum is generally used as a protein supplement in culture media for human in vitro fertilization (IVF). To determine the effects of serum supplementation on the development of preimplantation embryos, one-cell and two-cell mouse embryos fertilized in vitro and in vivo were cultured in BWW medium supplemented with preovulatory patient serum (PS), human fetal cord serum (HCS), or bovine serum albumin (BSA). The rates of fertilization and development of embryos fertilized in vitro and in vivo were suppressed when the embryos were cultured with PS or HCS as compared with BSA. In the medium supplemented with HCS, growth of in vitro fertilized embryos was retarded at an earlier embryonic stage than was the case with embryos fertilized in vivo. These results suggest that the in vitro fertilized embryo culture model is more sensitive to culture conditions than is the in vivo fertilized embryo culture model. Incorporations of [3H]uridine and [3H]leucine were measured using embryos that developed to the blastocyst stage in a medium supplemented with HCS or BSA. Blastocysts cultured with HCS showed significantly higher incorporations of [3H]uridine and [3H]leucine than those cultured with BSA. While serum supplementation in culture media has adverse effects on embryo cleavage, it augments synthesis of RNA and protein.

The serum insulin-like growth factor-II/mannose-6-phosphate receptor in normal and diabetic pregnancy

The extracellular domain of the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man-6-P) receptor is present in the circulation of several species including man. The purpose of the present study was to establish whether this truncated receptor is present in higher concentrations in fetal sera compared with adult sera and whether the metabolic status of the individual alters serum concentrations of this protein. Nondiabetic and diabetic pregnant women were studied throughout gestation, and at term fetal cord sera were obtained. Levels of IGF-I increased throughout pregnancy in normal and diabetic women. IGF-II levels significantly increased during the third trimester in both groups and levels of IGF-I and IGF-II were significantly elevated in fetal cord samples from diabetic women only. Serum samples were gel-filtered on Sephadex G-200, and column fractions were assayed for binding of radiolabeled IGF-II and IGF-I. There was specific binding (SB) of IGF-II in the void volume fractions in all samplex examined. Normal women had 3% ± 0.5% SB, whereas in cord sera SB was 5% ± 0.7% and in pregnant sera 10% ± 2%. There was no difference in SB in fetal cord or pregnant samples from normal and diabetic women. In addition, there was a peak of binding activity of both IGF-I and -II in γ-globulin and postalbumin fractions of the columns in pregnant and nonpregnant women, but only in postalbumin fractions in fetal cord samples. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of Sephadex G-200 void volume pools from all groups; a band of approximately 210 kd (without dithiothreitol) was seen. The band appeared to be more intense in pregnant women and to be increased in both the fetal cord samples and maternal samples from diabetic women. Based on binding and Western blotting, fetal cord samples had more receptor than nonpregnant normal women. When fetal cord, maternal, and adult nonpregnant sera Sephadex G-200 pools were assayed for IGF-II content by radioreceptor assay (RRA) after gel filtration in acid, only 1% to 2% of circulating IGF-II was associated with the truncated IGF-II/Man-6-P receptor in all cases. These data demonstrate that the extracellular domain (ecto-domain) of the IGF-II/Man-6-P receptor circulates in fetal cord serum and is elevated compared with that in normal adult women. Maternal concentration of the receptor is also increased in pregnancy. The ecto-domain of the IGF-II/Man-6-P receptor is not a significant carrier of the peptide and may reflect the increased levels of the peptide during pregnancy.

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction.

We studied the effect of media composition on sperm capacitation, using Biggers-Whitten-Whittingham (BWW) medium, Ham's-F10 and a modified Tyrode's medium (HSM) supplemented with bovine serum albumin (BSA) or fetal cord serum (FCS). We evaluated the effect of chemical environment and protein supplementation on the sperm motion parameters of curvilinear velocity and linearity, and on the ability of incubated spermatozoa to undergo follicular fluid induced acrosome reaction. Neither chemical composition nor protein supplementation of capacitation media greatly affected motion parameters after 2 h incubation. Furthermore, chemical composition had only a small effect on the ability of spermatozoa to undergo the acrosome reaction upon exposure to follicular fluid. A higher proportion of spermatozoa underwent acrosome reaction after incubation in HSM (8% control (C); 28% follicular fluid) than in BWW (8% C, 17% follicular fluid) or Ham's F-10 (6% C, 19% follicular fluid). By contrast, protein source proved critical in determining acrosome reaction inducibility. Spermatozoa incubated in BSA-supplemented media showed a 4-fold increase in acrosomal discharge when exposed to follicular fluid (6% C, 22% follicular fluid) compared to controls while spermatozoa incubated in FCS were unable to undergo acrosome reaction (6% C, 6% follicular fluid). Simultaneous addition of FCS to BSA capacitation medium blocked acrosome reaction inducibility and the late addition of BSA, after sperm incubation in FCS, did not facilitate acrosome reaction. We propose that an inhibitor of sperm capacitation is present in FCS and therefore, the selection of optimum incubation conditions for spermatozoa may be of critical importance when evaluating or treating infertile patients.

Comparative Use of Immunological Methods & Ligand Blotting of Insulin-like Growth Factor Binding Proteins in Serum and Other Biological Fluids

There is controversy as to which methodology most accurately detects the Insulin-like growth factor binding proteins (IGFBPs). We have compared immunological and ligand binding techniques to assess the IGFBPs in serum from non-pregnant women and women in late pregnancy, fetal cord serum and amniotic fluid. A specific radioimmunoassay for IGFBP-3 measured mean levels of 3.5 and 3.7μg/ml for non-pregnancy and pregnancy serum, respectively, 0.7μg/ml for fetal cord serum and 5.1μg/ml for amniotic fluid. In contrast, Western-ligand blot of fluid showed IGFBP-3 as a 40 kDa doublet in non-pregnancy serum and fetal cord serum, unlike pregnancy serum or amniotic fluid, where IGFBP-3 was undetectable by standard blot analysis. However, analysis of fluids at 25-50 times more volume showed that IGFBP-3 was detectable as a 40 kDa doublet in pregnancy serum and amniotic fluid. The molecular mass of immunoreactive IGFBP-3 was examined by Western-immunoblot, probed with antibody α IGFBP-31l. As in Westem-ligand blot, intact IGFBP-3 (40 kDa) was easily detected in non-pregnancy serum and fetal cord serum, but required greater volumes of sample for detection in pregnancy serum and amniotic fluid. In addition, smaller immunoreactive fragments were found in all fluids: a faint 27 and 22 kDa band was found in non-pregnancy serum and fetal cord serum, with more dominant bands of 31-27, 26, 25, 24 and 22 kDa in pregnancy serum and 31-26, 25, 22, 19-17 kDa in amniotic fluid. To address the obvious discrepancies between ligand and immunological analysis, samples were assessed for IGFBP-3 protease activity. Non-pregnancy serum proteolyzed 14% of the substrate, pregnancy serum 61%, fetal cord serum 33%, and amniotic fluid 50-61%. Thus, fluids with apparently low IGFBP-3 by Western-ligand blot, but high IGFBP-3 by RIA, showed high levels of protease activity. Previously, IGFBP-proteases have been shown to degrade IGFBP to smaller fragments with a reduced affinity for IGF. We conclude that the discrepancy between ligand binding techniques and immunological techniques can be explained, at least in part, by the presence of IGFBP-protease activity.

Simultaneous ectopic pregnancy with intra-uterine gestation after in vitro fertilization and embryo transfer

A case of combined intra-uterine and tubal ectopic pregnancy is described following in vitro fertilization and the transfer of two four-cell and one two-cell embryos. This phenomenon is know to be related to ovarian stimulation by gonadotropin therapy, and there is an increased risk with tubal disease. Techniques applied at the time of embryo transfer, the use of culture medium with 50% fetal cord serum to convey the embryos to the uterus, the catheterization method, and the position of the patient during transfer are presented. The risk of multiple pregnancies and combined intra-uterine and ectopic gestations increases with numbers of transfers and large volume of transfer medium. We would therefore recommend that after IVF-ET treatment in women with tubal disease, intensive care should be taken in the early follow-up period to rule out the possibility of ectopic pregnancy. In this case, a viable ongoing intra-uterine pregnancy was confirmed after surgery for right ampullary ectopic pregnancy. And a 2,925 g male in excellent condition was delivered by Cesarean section without complications.

Effects of human sera and human serum albumin on mouse embryo culture.

Human proteins normally used to supplement human in vitro fertilization-embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.

Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program

This study describes the results with immature human follicular oocytes harvested from unstimulated ovaries, matured in vitro, fertilized, and transferred to an agonadal recipient. Two hundred seventy immature oocytes were aspirated from 23 ovaries removed for various gynecological indications from August 1988 to October 1989. The numbers of follicular oocytes collected from ovaries were compared by patients' ages and the stages of menstrual cycle. Immature oocytes in vitro were incubated with either mature follicular fluid (FF) or fetal cord serum (FCS). The maturation rate in the mature FF group was 55.8%, significantly higher than the 35.9% in the FCS group. In addition, mature FF group was shown to provide a significantly higher fertilization rate than the FCS group (81.0% versus 31.6%). More fertilized eggs developed into normal embryos in the nonstimulated cycle group than in stimulated cycles with routine treatment. Finally, five embryos were transferred to a woman with premature ovarian failure on day 18 of a steroid replacement cycle. She subsequently delivered healthy triplet girls. These results suggest that in vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in a donor oocyte program after in vitro fertilization.

The incidence of human immunodeficiency virus (HIV) in fetal cord samples utilized as serum supplements for in vitro fertilization

Prior to initiating routine fetal cord serum (FCS) supplementation in our in vitro laboratory, the incidence of HIV in 306 random fetal cord samples obtained at the Labor and Delivery Unit of the Johns Hopkins Hospital from July 1985 to January 1988 was determined from a cross-sectional patient sample. Of 306 samples, 3 (0.98%) were positive for HIV, and confirmed by Western blot analysis, enzyme immunoassay (ELISA), a prevalence significantly higher than a national sample (0.012%). The use of FCS in this setting was determined to be an unacceptable risk to patients undergoing in vitro fertilization. The 90-day quarantine period for verification of HIV seronegativity applied to semen donors may not be applicable to FCS sampling. Patient risk may also increase with pooling of FCS samples prior to HIV testing.

Human follicular fluid stimulates hyperactivated motility in human sperm

Since human follicular fluid (FF) is known to enhance the acrosome reaction and capacitation, we investigated whether hyperactivated motility is stimulated by FF. Follicular fluid-treated sperm exhibited a threefold increase in hyperactivation compared with the controls. The use of fetal cord serum in the medium, instead of bovine serum albumin, supported the same high levels of hyperactivation, although the peak occurred at 3 hours rather than 5 hours of capacitation. When sperm were treated with a steroid-rich fraction of the FF, hyperactivation was stimulated to the same degree as with whole FF. In contrast, no stimulation occurred when sperm were treated with a FF fraction stripped of steroids. The FF enhancement of hyperactivation in vitro could augment the fertilizing capacity of subfertile sperm samples, providing also a glimpse of possible in vivo events as sperm traverse the FF-laden cumulus oophorus.

Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization

Patient or fetal cord serum is commonly used as a protein supplement to culture media used in in-vitro fertilization (IVF). To eliminate the variability and possible hazards related to the use of human serum, a well-defined protein supplement, Albuminar-20 (Armour Pharmaceutical Cy) was evaluated as a substitute for serum. Prior to its application in the human, Earle's culture media supplemented with 0.5% (w/v) bovine serum albumin, 8% (v/v) decomplemented patient serum or 2.25% (v/v) Albuminar-20 were compared in a mouse bioassay. For the three different conditions, the percentages of blastocysts formed after 120 h in-vitro culture were respectively 91.2, 85.2 and 87.8% (NS). In the human IVF, a controlled comparison was performed from October to December 1988, between Earle's medium supplemented with patients' serum or Albuminar-20. When oocytes and spermatozoa were cultured in these two media, the fertilization rates were similar, 58.9% in human serum versus 59.4% in Albuminar-20. After further culture, the morphological quality of the cleaved embryos was better in the embryos cultured in Albuminar-20. The higher pregnancy rate in Albuminar-20 was correlated with the better morphological appearance of the embryos and their more advanced cleavage stage at the time of transfer. Therefore, Albuminar-20 can be considered as a suitable protein supplement in human IVF.

Development of two-cell mouse embryos in protein-free and protein-supplemented media

Two-cell mouse embryos from CFW (Swiss-Webster) mice were cultured to the blastocyst stage in Ham's F10, Whittingham's T6, or human tubal fluid medium. Media were used without any protein supplements or were supplemented with human maternal serum, human fetal cord serum, or human serum albumin. Blastocysts were transferred to modified Eagle's basal medium supplemented with 10% fetal bovine serum for postblastocyst development. Blastocyst and postblastocyst development was depressed among embryos cultured during the preimplantation stage in protein-free Whittingham's T6 and human tubal fluid media compared with embryos cultured in protein-free Ham's F10 medium. This advantage of Ham's F10 disappeared when amino acids and vitamins were added to the other two media. Whittingham's T6 and human tubal fluid supplemented with human serum albumin, human maternal serum, or human fetal cord serum also supported excellent embryo development. When supplemented with protein, Ham's F10 was the poorest of the media in supporting embryo development. Although these results suggest that Ham's F10 is not the best medium for culture of mouse embryos, there is need for caution in extrapolating results from the mouse to the human.

The neuropeptide histidyl proline diketopiperazine throughout human pregnancy: An inverse correlation with amniotic fluid prolactin

Histidyl proline diketopiperazine values have been established in human amniotic fluid (n = 81) and maternal serum (n = 36) throughout gestation (10 to 42 weeks). Newborn cord serum (n = 10) and first-voided fetal urine (n = 10) levels were also documented. These measurements reveal increasing amniotic fluid levels with term gestation values (15,551 pg/ml) nearly thirteen-fold higher than maternal serum concentrations (1150 pg/ml). Corresponding fetal urine and cord serum concentrations were 16,781 and 2160 pg/ml, respectively. The amniotic fluid values are not influenced by fetal sex or maternal labor, nor do they correlate with amniotic fluid a-fetoprotein levels. However, there is a significant inverse correlation (r = -0.628; p<0.0001) between amniotic fluid prolactin and histidyl proline diketopiperazine after midgestation. The hypothesis that histidyl proline diketopiperazine may be a regulatory peptide for decidual prolactin production was tested by culturing term decidua in the presence of varying concentrations of histidyl proline diketopiperazine, but no inhibitory effect was observed. Decidual cultures did not produce measurable amounts of histidyl proline diketopiperazine. It is suggested that amniotic fluid histidyl proline diketopiperazine is derived from fetal urine. (AM J OesTET GYNECOL 1990;162:740-5.)

Effect of different procedures of semen preparation on antibody-coated spermatozoa and immunological infertility

To assess whether procedures of semen preparation can reduce the proportion of antibody-coated spermatozoa, semen samples with positive direct mixed antiglobulin reaction (MAR) were washed in media supplemented with 10% or 50% fetal cord serum (FCS). Washing reduced the MAR to a negative level, but the MAR was identical to that in the native semen when spermatozoa were resuspended in serum-free medium. Donor spermatozoa, recovered after swim-up in media supplemented with 10% or 50% FCS or after passage through a column with 7.5% human serum albumin (HSA), were incubated in serum samples with both agglutinating and cytotoxic antisperm antibodies. Cytotoxic activity was significantly reduced against sperm filtered over the albumin column.

Evaluation of a synthetic serum substitute to replace fetal cord serum for human oocyte fertilization and embryo growth in vitro

A comparison was made between a serum substitute. UltroSer G (Gibco, Ghent, Belgium) (2%) (medium B) and 10% human fetal cord serum (medium A), as regards their ability to support 1-cell and 2-cell mice embryo development in vitro. Sixty percent and 56% of the 1-cell embryos reached the expanded blastocyst stage when cultured in media A and B, respectively. Eighty-four percent and 88% of 2-cell embryos reached the expanded blastocyst stage when cultured in media A and B, respectively. A prospective randomized study was then performed to evaluate this synthetic serum substitute in human in vitro fertilization. Among 141 ovum pick-up (OPU), oocytes retrieved in 74 cases were processed in medium A and oocytes retrieved in 67 others in medium B. In media A and B, the fertilization rate was 67% and 44.3% respectively, and the pregnancy rate/OPU 23% and 9%, respectively. The pregnancy rate/transfer was 28.8% and 12.2% respectively, and the implantation rate/transferred embryo 9.5% and 4.2%. In the human sperm survival assay, the vitality and residual motility after 24 hours of incubation were significantly lower in medium B. In conclusion, UltroSer G successfully sustained the development in vitro of mouse embyros. However in human, it reduced sperm survival, oocyte fertilization, and embryo viability.

The use of human follicular fluid in gamete intra-Fallopian transfer

We undertook a prospective study to compare our gamete intra-Fallopian transfer (GIFT) procedure with or without the use of human follicular fluid (FF) as a constituent for the final spermatozoal suspension and as the tubal transfer medium for both eggs and spermatozoa. We routinely perform an intrauterine and intracervical insemination (IUI and ICI) following GIFT, and FF or culture medium was used accordingly as a constituent in this spermatozoal suspension also. When FF was used (26 cycles), clear FF taken from the first egg-bearing follicle was sterilized by micropore filtration, gassed with 5% CO2 in air and warmed to 37 degrees C. This FF was then used to dilute the spermatozoal suspension (50:50, v/v) for both tubal, uterine and cervical inseminations at least 30 min before transfer, and all transferable eggs were placed into this FF before transfer. Alternatively (30 control cycles), eggs and spermatozoa were prepared and transferred in Earle's medium supplemented with 10% pooled fetal cord serum. The FF and control patient groups were relatively homogeneous, with no statistically significant differences in ovarian response, oocyte retrieval or transfer or seminal profiles. The outcome of the GIFT procedures using FF or culture medium showed no significant advantage of the use of FF. The clinical pregnancy rate was similar in both groups: 50% (15/30) control; 46.2% (12/26) FF.

Insulin and Insulin-Like Growth Factor-I Stimulate the 3α-Hydroxysteroid Dehydrogenase Activity of Human Placental Cytotrophoblasts*

The placenta is the primary source of progesterone during pregnancy. Because pregnant diabetic women are reported to have higher serum progesterone levels than nondiabetic pregnant women, we studied the roles of insulin and insulin-like growth factor-I (IGF-I) in the regulation of human cytotrophoblastic 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity. Incubation of cytotrophoblasts with insulin or IGF-I for 24 h significantly increased the ability of these cells to convert pregnenolone to progesterone by 75.8 +/- 16.5% (+/- SE) and 65.4 +/- 12.7%, respectively. Treatment with either insulin or IGF-I did not alter cytotrophoblastic production of 20 alpha-hydroxypregn-4-en-3-one (the primary metabolite of progesterone), thus demonstrating that these peptides increased progesterone synthesis (i.e. 3 beta HSD activity) rather than decreased progesterone catabolism. Insulin and IGF-I stimulated 3 beta HSD activity at concentrations as low as 50 and 10 ng/ml, respectively. Insulin- and IGF-I-stimulated 3 beta HSD activities were completely inhibited by concurrent treatment with either actinomycin-D or cycloheximide, suggesting that new mRNA and protein synthesis are required for these peptides to exert their effects. Blockade of the IGF-I receptor of cytotrophoblasts with alpha IR-3, a monoclonal anti-IGF-I receptor antibody, prevented the stimulation of 3 beta HSD activity by IGF-I, but did not alter insulin's stimulatory effect. Thus, the two hormones can each stimulate 3 beta HSD activity via activation of their respective receptors. These studies indicate that insulin and IGF-I can regulate human cytotrophoblastic 3 beta HSD activity in vitro. Since pregnant diabetic women manifest peripheral hyperinsulinemia, and IGF-I levels in fetal cord sera from diabetic pregnancies may be elevated, these observations may help explain the elevated serum progesterone levels associated with pregnancy in the diabetic patient.

Hormonal and basement membrane markers for immunoidentification of cultured human trophoblast cells

Improvement of human chorionic villi cells in vitro culture has been obtained by supplementing the medium with 30% human fetal cord serum, instead of fetal calf serum. Expression of human chorionic gonadotropin, estrogen and progesterone receptors, laminin, laminin receptors, and type IV collagen has been studied on first subculture passages (4–7 weeks) by immunoperoxidase method. A minority of the cultured cells were positive for estrogen receptors, the majority were positive for progesterone receptors, while all cells were negative for human chorionic gonadotropin. Cultured cells showed variable positive immunostaining for basement membrane markers like laminin, and type IV collagen, and for laminin receptors. Detection of both progesterone receptors and laminin, or type IV collagen, excluded fibroblast contamination and could then be useful for quick identification of cultured trophoblast cells.

Conformational changes in rodent and human α-fetoprotein: Influence of fatty acids

Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.

Cell-free human amniotic fluid as culture medium for mouse and human embryos

AbstractDifferences between murine and human preimplantation embryonic growth in cell-free human amniotic fluid and medium F -10 supplemented with human fetal cord serum were assessed. Two-cell mouse embryos cultured in human amniotic fluid developed more consistently to the expanded blastocyst stage than those cultured in F-10. No significant difference in human preimplantation embryo cleavage was observed between the two culture conditions. However, amniotic fluid was significantly worse than F-10 +human serum in its ability to maintain embryos that could establish pregnancy after transfer.

Freezing-thawing behaviour and cell membrane ultrastructure of mouse embryos pre-cultured in B2-Menezo medium before cryopreservation.

Mouse embryos were pre-cultured from the 2-cell to the 8-cell stage inserum-free B2-Menezo medium and in B2-Menezo medium with 15% fetal cord serum before cryopreservation with a slow freezing-thawing method (-0.3 degree C/min in 1.5 M DMSO). Viability after freezing-thawing was tested by continuing in-vitro culture and evaluating the percentage of embryos developing to the blastocyst stage. An extreme decrease in viability after freezing and thawing was found for the embryos pre-cultured in serum-free B2-Menezo medium compared with the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum (12 versus 81% blastocysts). Ultra-thin sections of the pre-cultured embryos and of freshly collected controls were prepared for transmission electron microscopy before and after the freezing-thawing procedure. A drastic decrease in the number of microvilli on the cell surface and a high degree of cell damage after freezing and thawing was observed in the embryos pre-cultured in serum-free B2-Menezo medium. In contrast a high number of microvilli and an intact cell structure after cryopreservation was observed in the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum and in the non-pre-cultured controls. It is suggested that the smaller cell surface and the resulting lower permeability of the cell membrane in the embryos with few microvilli are due to sub-optimal culture conditions and are the main reasons for the extremely decreased viability after freezing and thawing.