Caffeine and other predictors of bone density among pre- and perimenopausal women

Cryopreservation has proved to be a highly successful method for long-term storage of viable embryos. The objective of this study on rat blastocysts was to define conditions for their cryopreservation. Three cryoprotectants, dimethyl sulfoxide, glycerol, and propanediol/sucrose, were compared in two cooling programs (to -30 or -80 degrees C) and two thawing protocols. The cooling was followed by immersion in liquid nitrogen. Programmed thawing was at the rate of 8 degrees C per minute; fast thawing consisted of direct exposure of the frozen embryos to the ambient laboratory temperature. The survival after the freeze/thaw was assessed from the post-thaw embryo morphology and ability to develop into apparently normal offspring in uteri of foster mothers (embryonic survival). The best method for preservation of rat blastocysts proved to be programmed cooling to -80 degrees C followed by fast thawing with glycerol as cryoprotectant (embryonic survival of 28.1%). In all the experimental groups, the proportion of embryos with good to excellent preservation of morphology was high. With dimethyl sulfoxide, after programmed cooling to -80 degrees C, embryonic survival was 9.9% (programmed thawing) and 17.5% (fast thawing). No embryos survived after programmed cooling to -30 degrees C. However, when the cryoprotectant was propanediol/sucrose, no difference was observed between programmed cooling to -80 degrees C with either method of thawing and programmed cooling to -30 degrees C and fast thawing (12.3, 6.2, and 8.0%, respectively).     

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61% in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts.     

Development of embryos produced by intracytoplasmic sperm injection of cat oocytes

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.     

Effect of level of dietary energy and protein on embryo survival and progesterone production on day eight of pregnancy in Rasa Aragonesa ewes

The aim of this experiment was to investigate the effects of dietary protein and energy on ovulation rate and embryo survival to day 8 of pregnancy, and the associated concentrations of progesterone in jugular, ovarian and uterine veins, in a Spanish breed of sheep. In mid-October, three groups of ewes were fed to provide 1.5 x (H; n = 9), 0.5 x (L; n = 12) or 0.5 x plus 7.44 g CP/MJ ME (LP; n = 8) energy requirements for maintenance of live weight from day -14 relative to a synchronized mating on day 0. A significant effect of nutrition on ovulation rate was observed (H: 2.22 +/- 0.16; L: 1.50 +/- 0.16; LP: 1.88 +/- 0.12 corpora lutea; P < 0.05). Mean LH and progesterone concentrations were affected by nutrition on day 7, L ewes showing the highest mean LH level (P < 0.01), while H ewes presented the lowest mean LH concentration and the highest mean plasma progesterone concentration (P < 0.01). Laparotomies were performed on six animals of each group on day 8 to determine the effect of nutrition on embryo development. A significantly higher percentage of embryos recovered from L and LP ewes presented an earlier stage of development (morulae or early blastocysts) (P < 0.001), while 100% embryos of H ewes were expanded blastocysts. The ratio expanded blastocysts/corpora lutea was significantly higher in H ewes (0.86) when compared with L and LP groups together (0.57; P < 0.05). Mean progesterone concentration in the ovarian vein was 800-fold higher than mean jugular venous levels with no differences between groups. Samples from ovarian veins contralateral to corpus luteum-bearing ovaries showed mean progesterone concentrations significantly lower than samples opposite to corpus luteum (ipsilateral: 1037.84 +/- 138.45; contralateral: 30.4 +/- 11.22 ng/ml; P < 0.001). Mean progesterone concentration in the uterine vein was approximately 30-fold higher than in jugular and similar in both uterine horns and treatments. No effect of nutrition on pregnancy rate was observed (H: 89%; L: 92%; LP: 100%). These results suggest that neither dietary energy nor protein are able to modify pregnancy rate or progesterone concentrations in ovarian and uterine veins eight days after mating. However, the delay in embryo development observed in the embryos collected from L and LP ewes may give rise to compromised embryo growth and development some days later.     

Aggressive behavior of psychiatric patients among geriatric population

The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In Experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following: 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P < 0.01) in MK-1 + BSA (74.4%), MK-1 + FCS (56.1%), and M-199 + FCS (51.4%) than in MK-1 + HS (94.7%). A greater (P < 0.01) percentage of blastocysts was obtained in MK-1 + HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P < 0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulae cultured in MK-1 + BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1 + HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET.     

Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol

Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.     

Reproductive wastage in the androstenedione-immune ewe

An experiment was carried out using 320 adult Merino ewes to examine the effects of immunization against an androstenedione human serum albumin conjugate (Fecundin) on ovulation rate, fertilization rate and embryo viability at days 2, 9 and 13-14 after fertilization. The ovulation rate of immunized ewes (2.19 +/- 0.06) was greater (P < 0.001) than that of control ewes (1.43 +/- 0.04). The recovery rate of embryos or of unfertilized oocytes on day 2 was reduced in immunized ewes, but fertilization rate of recovered oocytes was unaffected by immunization. The mean number of normal embryos per ewe pregnant (prolificacy) was higher and the proportion of ewes pregnant (fertility) was lower in immunized than in the control ewes. The distribution of the number of cells per embryo showed no differences in developmental age over the period of sampling, the majority of embryos at this time being at the two- to four-cell stage of development. At day 9 of pregnancy, blastocyst recovery rates were lower in immunized than in control ewes. About 90% of blastocysts recovered were developing normally in control ewes compared with 64% in immunized ewes. The majority of blastocysts recovered on day 9 had hatched from the zona pellucida prior to recovery (mean values were 94.2% and 87.8% for control and immunized groups, respectively). In control ewes single blastocysts were larger than twin blastocysts, but for the immunized ewes this difference was not significant. Both single blastocysts (P < 0.01) and twin blastocysts (P < 0.05) from immunized ewes were smaller than those from control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence?

The success of intracytoplasmic sperm injection (ICSI) warrants further study on the role of paternal factors in early human embryogenesis. To investigate whether poor sperm parameters can influence embryo development, we examined the development of ICSI-fertilized embryos to the blastocyst stage. We present results of blastocyst development from supernumerary ICSI embryos after co-culture on monkey kidney epithelial cells. In addition, we compare the development of supernumerary embryos to the blastocyst stage after ICSI and in-vitro fertilization (IVF). Of 168 supernumerary ICSI embryos, 45 (26.8%) developed to blastocysts. Sperm concentration and morphology did not influence blastocyst development. In contrast, blastocysts arose from spermatozoa that had a significantly higher (P = 0.015) forward progressive motility compared with spermatozoa from those patients who failed to produce blastocysts (42.7% versus 28.2%, respectively). Overall the rate of embryo development to the blastocyst stage after ICSI was lower (26.8%) than that after IVF (47.3%). When the rate of blastocyst development was calculated for patients with three or more supernumerary embryos, it remained significantly higher for the IVF patients than for the ICSI patients (45.6% versus 30.0%). There was no significant difference in the mean cell number and quality of the supernumerary embryos between the IVF and ICSI patients. This study confirms previous reports that have postulated that abnormal spermatozoa may manifest a negative paternal effect on preimplantation embryo development.     

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

Premature progesterone elevation spares blastulation but not pregnancy rates in in vitro fertilization with coculture

OBJECTIVE: To clarify whether embryo development to the blastocyst stage may be affected by premature P elevation during controlled ovarian hyperstimulation (COH) for IVF-ET with embryo coculture. DESIGN: Retrospective study. SETTING: Tertiary care infertility center. PATIENT(S): One hundred thirty-one women undergoing 153 IVF-ET cycles with embryo coculture. INTERVENTION(S): Patients underwent COH with GnRH agonist and hMG. Embryos were cocultured up to the blastocyst stage. According to plasma P levels on the day of hCG, two groups were defined: low P (P < or = 0.9 ng/mL; conversion factor to SI unit, 3.180) and high P (P > 0.9 ng/mL). MAIN OUTCOME MEASURE(S): Blastulation (number of blastocysts/number of noncavitating embryos x 100) and pregnancy rates (PRs). RESULT(S): Blastulation rates were similar in the low and high P groups (51% and 48%, respectively). Moreover, patients included in the high P groups achieved significantly lower clinical and ongoing PRs (12% versus 29% and 7% versus 25%, respectively). CONCLUSION(S): The lack of difference in blastulation rates between the groups further supports the hypothesis that premature P elevation does not alter oocyte and embryo quality. Hence, the observed decrease in PRs is likely to reflect impaired endometrial receptivity in the high P group.     

Monoclonal antibodies to mammalian heat shock proteins impair mouse embryo development in vitro

Two-cell mouse embryos (B6D2F1) were cultured in the presence or absence of 100 microg/ml monoclonal antibodies specific for the mammalian 60 kDa (HSP60), 70 kDa (HSP70) and 90 kDa (HSP90) heat shock proteins. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. At day 3, only 29% (22/75) of the embryos cultured with anti-HSP60 antibody developed to the blastocyst stage (P < 0.0001) as compared to 67% (31/46) of the embryos cultured with anti-HSP70, 72% (43/60) cultured with anti-HSP90, and 79% (49/62) in medium plus mouse IgG1. By day 5, hatched embryos were present in 28% (13/ 46) of the cultures containing anti-HSP70 (P < 0.0001), as opposed to 57% (34/60) containing anti-HSP90 and 73% (45/62) containing IgG1. At day 7, outgrown trophoblasts were observed in 9% (4/46) of cultures containing anti-HSP70 (P < 0.0001), 45% (27/60) containing anti-HSP90 (P < 0.01) and 66% (41/62) cultured in medium plus IgG1. Antibodies to different heat shock proteins exerted a detrimental effect on mouse embryo development at unique development stages. Immune sensitization to heat shock proteins may be a cause of reproductive failure.     

Succinate and malate improve development of hamster eight-cell embryos in vitro: confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% +/- 3.9 vs. 54.5% +/- 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% +/- 7.2), but not pyruvate (85.8% +/- 6.2) or glutamine (84.1% +/- 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% +/- 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 +/- 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 +/- 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 +/- 1.1) or malate (24.7 +/- 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (+/- 4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% +/- 2.0) or blastocysts (28.9% +/- 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium.     

Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers

The purpose of this study was to devise an embryo score to predict the likelihood of successful implantation after in-vitro fertilization (IVF). Unlike most studies dealing with the influence of embryo stage and morphology on pregnancy, our study was based on single rather than multiple embryo transfers. A total of 957 single embryo transfers were carried out. No delivery was obtained after any of the 99 transfers using 1-cell embryos or embryos obtained after delayed fertilization. In the remaining 858 transfers, the embryos had cleaved. Higher pregnancy rates were obtained with embryos displaying no irregular cells (11.7 versus 6.9%; P < 0.01) and embryos displaying no fragmentation (11.5 versus 8.1%; P < 0.05). The 4-cell embryos implanted 2-fold more often than embryos with more or less cells (15.6 versus 7.4%; P < 0.01). Based on these observations, we devised a 4-point embryo score in which embryos are assigned 1 point each if they (i) are cleaved, (ii) present no fragmentation, (iii) display no irregularities, and (iv) have four cells. Both pregnancy rate and take home baby rate were significantly correlated with embryo score. Each point of this score corresponds to a 4% increase in pregnancy rate. Interestingly, pregnancy rate was significantly lower in women aged > 38 years (8.2 versus 11.4%; P < 0.05), even though embryo quality was similar regardless of age. Single embryo transfer allowed us to define a simple and useful embryo score to choose the best embryo for transfer to optimize IVF and embryo transfer outcome. The use of this embryo score could decrease multiple pregnancies after multiple embryo transfers.     

Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.     

A method for endoscopic embryo collection and transfer in the rabbit

Thirty-two rabbits were used for endoscopic embryo collection and transfer. For embryo collection midventral laparoscopy and transcervical endoscopy were combined for orthograd flushing of the oviducts and uterine horns. Transfer was performed transcervically under laparoscopic control. The mean number of corpora lutea counted in nine donors was 13.3 +/- 8.4. A total of 72 morulae/blastocysts were obtained. No embryos were recovered when only the uterine horns were flushed. 291 embryos were transcervically transferred to 23 recipients which resulted in 10 pregnant animals at day 12. Two of three slaughtered recipients showed together 4 implantation sites. Four animals delivered 12 pups.     

Vertebral artery trauma

The developmental consequences of paternal exposure to acrylamide (50 mg/kg i.p. for 5 days) were assessed in preimplantation embryos. There was a significant increase in the proportion of morphologically abnormal embryos after postmeiotic treatment during spermatogenesis (88.7% vs. 14.8% in control). Abnormal embryos had an average of 1.8 +/- 3.5 cells and > 80% had at least one fragmented nucleus. In addition, morphologically normal embryos were significantly delayed (34.3 +/- 12.8 cells per embryo vs. 57.6 +/- 15.7 in control, P < 0.001). Acrylamide caused 10- and 20-fold increases in frequencies of cells with micronuclei (MN) in morphologically normal and abnormal embryos, respectively (41 and 93 MN per 1,000 cells). Both centromere-negative (MN-) and centromere-positive (MN+) were induced. Nuclei of abnormal embryos were significantly larger (900 microm2 vs. 250 microm2) than controls. In addition, MN of abnormal embryos were larger than those of normal embryos (21.2 microm2 vs. 6.5 microm2, P < 0.01). Among control embryos, MN+ were significantly larger than MN- (P < 0.05). These findings suggest that the preimplantation embryo is a sensitive indicator of paternally transmitted effects on early development. Multiple mechanisms appear to be involved, including cytogenetic damage, proliferation arrest/delay, and fertilization failure. Future studies are needed to establish how induced cytological defects in preimplantation embryos contribute to birth defects and other postimplantation abnormalities.     

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

Platelet activating factor (PAF) was administered to female mice in order to investigate its effect on ovulation rate and on oocyte quality including their in-vitro embryonic development, implantation and uterine receptivity. In experiment 1, 4-week-old female mice were assigned to receive PAF or phosphate buffered saline for 4 consecutive days. On the second day of this treatment, pregnant mares' serum gonadotrophin was administered and human chorionic gonadotrophin (HCG) 48 h later, after which copulation occurred. Oocytes were collected on the following day and evaluated. The mean number of oocytes and zygotes (two pronuclear stage embryos) recovered from the PAF-treated group was not different from the control group (31 versus 27), but the proportion of zygotes was higher in PAF-treated group than in controls (83 versus 68%, P < 0.05, PAF versus controls). Although the rate of in-vitro first cleavage was not different in the two groups (82 versus 69% respectively), hatching was higher in the PAF-treated group than control mice (99 versus 83%, P < 0.01). In experiment 2, the in-vitro developed blastocysts from experiment 1 were transferred into the uterus of day 3 pseudopregnant PAF-treated or control recipients. Three different combinations of intrauterine transfer were performed; PAF embryo to control recipient (PAF-->C: n = 19), control embryo to PAF recipient (C-->PAF: n = 19), and control embryo to control recipient (C-->C: n = 22). Implantation and abortion were assessed on day 19 posttransfer. The implantation rate of C-->PAF (23.7%) was lower than C-->C (31.1%, P < 0.05), but was not different from PAF-->C (31.2%). Further, C-->PAF showed a higher abortion rate per embryo (29.6%) than PAF-->C (12.7%, P < 0.05), but was not different from C-->C (24.4%). In the present study, PAF administration enables females to produce oocytes with a higher potential for fertilization, in-vitro development and implantation, but has a detrimental effect on uterine receptivity to embryos.     

Long-term evaluation of implantation of fresh and cryopreserved human embryos following ovarian stimulation with buserelin acetate-human menopausal gonadotrophin (HMG) or clomiphene citrate-HMG

This study is a long-term evaluation of the total pregnancy potential of cohorts of fresh and cryopreserved sibling embryos from in-vitro fertilization (IVF) cycles stimulated with either the gonadotrophin-releasing hormone analogue buserelin (BUS) (long protocol) or clomiphene citrate (CC) both in combination with human menopausal gonadotrophin (HMG). Therefore a retrospective analysis was performed on patients who entered the IVF programme between January 1986 and July 1987 and who had triple embryo transfer in the collection cycle. Significantly more fertilized oocytes developed to good-quality embryos in the CC-HMG group (86.1%) than in the BUS-HMG group (80.8%). Transfer of the three morphologically best-looking embryos was performed in day 2 post-insemination in 106 CC-HMG and 80 BUS-HMG cycles. Supernumerary embryos were cultured for a further 24 h and multicellular embryos with up to 20% of fragments were frozen slowly with 1.5 M dimethylsulphoxide on day 3 post-insemination (162 embryos in CC-HMG cycles, 102 embryos in BUS- HMG cycles). Outcome was measured by embryo survival rate, embryo implantation rate and delivery rate in fresh and frozen embryo transfers. Delivery rates were 31.3 and 21.7% per fresh embryo transfer in BUS-HMG and CC- HMG cycles respectively. Fresh embryo implantation rates were significantly higher in collection cycles stimulated with BUS-HMG (17.9%) than in cycles stimulated with CC-HMG (11.3%). Implantation rates were significantly enhanced in embryos transferred in excess of one in cycles leading to pregnancy, perhaps indicative of higher embryo quality in BUS-HMG cycles. Almost all cryopreserved embryos have by now been thawed, so the contribution of frozen embryos to overall pregnancy rates can be evaluated. Overall morphological survival rates of frozen-thawed embryos have by now been thawed, so the contribution of frozen embryos to overall pregnancy rates can be evaluated Overall morphological survival rates of frozen-thawed embryos were similar for 140 embryos from CC-HMG cycles (50%) and 100 embryos from BUS-HMG cycles (46%). The percentage of fully intact embryos was, however, significantly lower in the BUS-HMG group (19%) than in the CC-HMG group (39.5%). Delivery rates were significantly lower following 30 transfers of frozen-thawed embryos from BUS-HMG-stimulated cycles (3.3%) than following 42 transfers of frozen-thawed embryos from CC-HMG cycles (19.1%). Embryo implantation rates were lower for frozen-thawed embryos from BUS-HMG cycles (2.3%) than from CC-HMG cycles (12.7%). Here we demonstrate that ovarian stimulation with the long protocol BUS-HMG instead of the CC-HMG protocol led to higher embryo implantation rates in collection cycles but to lower intact embryo survival rates and to lower embryo implantation rates for frozen sibling embryos. Despite the lower implantation rates with frozen embryos originating from the BUS-HMG protocol, there was no significant difference between total delivery rate per transfer from cycles stimulated with CC-HMG (30.2%) compared with BUS-HMG (33.8%).