Outcome comparison of in vitro fertilization treatment with highly purified subcutaneous follicle-stimulating hormone (Fertinex, a urofollitropin) versus intramuscular menotropins.

OBJECTIVE: The aim of this study was to investigate various outcome measures of stimulation with highly purified subcutaneous follicle-stimulating hormone (Fertinex, a urofollitropin) compared with first- and second-generation urinary human menopausal gonadotropin standards (Pergonal, Metrodin). STUDY DESIGN: Retrospective analysis was restricted to our most efficient in vitro fertilization age group (23-34 years). Data from Institute for Assisted Reproduction in vitro fertilization cycles 1 through 11 with Pergonal, Metrodin, or both were tabulated for hormonal values, oocyte quality, and embryo outcome as baseline data. Patients in cycles 12 through 13 were treated with Fertinex and Pergonal or Fertinex alone and then reviewed for the same parameters. RESULTS: Two hundred thirty-eight in vitro fertilization records with embryo transfer were analyzed. Clinical pregnancy rates per embryo transfer in an optimal age group were similar despite use of first- through third-generation urinary gonadotropin preparations: Pergonal and Metrodin, 67%; Metrodin, 64%; Fertinex and Pergonal, 62%; and Fertinex, 54%. There were no discernible differences in hormonal response, oocyte recovery, or embryonic growth. CONCLUSION: Administered subcutaneously, the third-generation urinary gonadotropin preparation Fertinex is effective in in vitro fertilization treatment in young women.

The importance of water quality for media preparation.

The variability in pregnancy rates achieved among in-vitro fertilization (IVF) clinics may be partially attributable to the disparate quality of the water used in the preparation of culture media. The removal of contaminants in the water is of paramount importance since water constitutes the predominant component in any media formulation. To assist in the selection, operation and maintenance of a water purification system, the level of contaminants must be carefully monitored. Conductivity and resistance are used to measure the purity of natural and ultrapure water respectively. Feed water is analysed by an assortment of direct chemical means to determine the necessary system filtration steps. In general, high quality water can be produced by combined reverse osmosis and electrodeionization of treated tap water. Processed water is supplied to an ultrapure water system to provide final polished water. A detailed water processing protocol is presented along with quality assurance guidelines to ensure the consistent production of high quality ultrapure water suitable for in-vitro human embryo culture.

The application of co-culture in assisted reproduction: 10 years of experience with human embryos.

Co-culture techniques using fetal bovine uterine fibroblasts or bovine oviductal epithelial cells have improved embryonic development prior to replacement in humans. In initial co-culture trials, embryo development and implantation rates increased after just 1 day in culture. The most overt characteristics noted following co-culture were improved blastomere development and characteristics, reduced fragmentation, and the appearance of swollen blastomeres. In addition, an increase in the incidence of zona thickness variation was detected. Improved development of polyspermic and supernumerary embryos to the blastocyst stage was noted in initial trials. Retrospective analysis indicated that certain patient subgroups benefit the most from co-culture. As a result, co-culture is now applied routinely to patients that have previously failed attempts at in-vitro fertilization (IVF) and/or have endocrine imbalances such as polycystic ovarian syndrome and elevated day 3 concentrations of follicle stimulating hormone (FSH). The use of co-culture prior to or following cryopreservation has also proven to be beneficial to human embryos. The proposed beneficial mechanisms thought to improve embryonic development include a secretory and/or a scavenging role. Evidence describing the postulated benefits is discussed.

Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients.

A study was conducted on patients who had attempted and failed previous in-vitro fertilization (IVF) procedures an average of 3.8 times following the application of assisted hatching with conventional culture systems. The aim of this investigation was to determine if addition of co-culture methodologies could reduce embryonic abnormalities and thus improve the prognosis for pregnancy. The study population consisted of 123 patients, subdivided into three patient categories. Previous IVF results from conventional culture were used to evaluate any potential benefits derived from the present co-culture application. Following co-culture, the rate of blastomere development was increased and the rate of fragmentation decreased. An increased rate of blastomere development was most noticeable in the patients aged < or = 39 years with no male factor as well as the intracytoplasmic sperm injection (ICSI) subgroup. Similarly, the rate of fragmentation was significantly reduced in the aforementioned subgroups. The most pronounced impact of co-culture was on pregnancy and implantation rates. The overall clinical and ongoing pregnancy rates were 38% (47/123) and 36% (44/123) respectively. The corresponding implantation rate was 17% (72/ 412) as shown by embryonic cardiac activity. The ongoing pregnancy rates in the < or = 39 years no male factor, > or = 40 years no male factor and ICSI no age limit patient subgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively. The results indicate that addition of co-culture to the IVF procedure for poor-prognosis patients may be advisable.

Blastocyst development in co-culture: development and morphological aspects.

A retrospective study was undertaken to determine if initial culture conditions and embryo quality had an effect on subsequent blastocyst development in co-culture for cryopreservation. The apparent effects of freeze-thawing on blastocysts at the ultrastructure level were also observed. On day 3 of culture, embryos were categorized into two groups based on their morphological attributes. Results suggest that the initial culture environment of embryos up to day 3 (5- to 8-cell stage) did not affect the subsequent rate of blastocyst formation in co-culture. However, the initial embryo quality had an impact on blastocyst formation and quality. On day 5.5, 90% (60/67) of the optimal quality embryos (six to eight blastomeres with minimal or no fragmentation on day 3) had attained the blastocyst stage, which was greater (P < 0.01) than the 55% (31/56) observed with the sub-optimal embryos (five to eight blastomeres with 30-50% fragmentation on day 3). Furthermore, 66% (44/67) of embryos initially graded as optimal were suitable for cryopreservation, which was greater (P < 0.01) than attained with embryos of lesser quality (22/56; 39%). At the ultrastructural level, the polarized distribution of plasma membrane microvilli was retained, as was the integrity of the nuclear membrane following thawing.

Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients.

Older patients and those who consistently return for embryo transfer but without implantation were studied to see if a combination of day 3 assisted hatching and co-culture (AH+CC) might be beneficial compared to assisted hatching alone (AH-alone). Female patients of > or = 38 years and couples who had previously failed to implant embryos three times or more were prospectively and randomly assigned to either an experimental or a control group. In the experimental group all embryos were co-cultured on partial monolayers of bovine oviductal epithelial cells for 2 days followed by assisted hatching by zona drilling (AH+CC). All control embryos were cultured by standard procedures until day 3 when they also underwent zona drilling prior to uterine transfer (AH-alone). With 50 cycles in each group there was unfortunately a marginal bias against the AH+CC group in that these patients had undergone a higher number of previous transfer cycles. There was a marginally lower percentage of fragmentation and a significantly higher degree of zona thickness variability in the AH+CC embryo group. Embryonic implantation was significantly increased (P < 0.05) in the AH+CC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantly higher (P < 0.05) initial pregnancy rate (52 versus 32%) in the AH+CC group, and a higher (not significant) viable pregnancy rate (38 versus 22%).

The combination of coculture and selective assisted hatching: results from their clinical application.

OBJECTIVE: To establish pregnancies using a combination of coculture and selective assisted hatching. DESIGN: Clinical application for a selected group of patients. Not a controlled study. SETTING: Private infertility practice. PATIENTS: Women with high basal FSH levels, ovulatory disorders, and multiple failed IVF attempts. MAIN OUTCOME MEASURES: Pregnancy and implantation rates. RESULTS: Of the 95 patients who had coculture and selective assisted hatching, 45 (47.0%) have an ongoing pregnancy with a 23.0% implantation rate. CONCLUSION: The combination of coculture and assisted hatching produced acceptable pregnancy and implantation rates within the selected patient population.

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

A study was undertaken to evaluate embryonic development and establish pregnancies with human embryos after in-vitro culture in two different systems. Treatment A consisted of culturing zygotes in serum-supplemented human tubal fluid culture medium (HTF). Treatment B consisted of culturing zygotes on a monolayer of bovine oviductal epithelial cells with HTF. At the time of embryo replacement, embryos in treatment B had 4.11 blastomeres present, which was greater (P < 0.05) than the 3.81 present for embryos in treatment A. In addition, the cellular fragmentation rate for treatment A embryos was 1.10, which was greater (P < 0.05) than the fragmentation rate of 0.38 for embryos within treatment B. The incidence of ongoing pregnancy was higher after replacement of co-cultured embryos (treatment B) (43%) than replacement of conventionally cultured embryos (treatment A) (29%). The implantation rate per embryo increased (P < 0.05) from 11.5 to 18.4% after co-culture. In treatment B the proportion of 'spare' embryos developing to expanded blastocysts was 58.5%, which was greater (P < 0.05) than the blastocyst development rate of 29.3% observed for embryos within treatment A.

U2 small nuclear RNA localization and expression during bovine preimplantation development.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.

Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo.

The sensitive technique of mRNA phenotyping with the reverse transcription-polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one-cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF-alpha) and platelet-derived growth factor (PDGF-A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF-beta 2 and insulin-like growth factor (IGF-II) and the receptors for PDGF-alpha, insulin, IGF-I, and IGF-II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two-cell stage. In contrast to the mouse embryo, IGF-I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight-cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.

Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P less than 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P less than 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P less than 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P less than 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation.

Zygotes from in vitro fertilization patients (n = 116) were randomly allocated to culture in either conventional plastic petri dishes or coculture on a monolayer of fetal bovine uterine fibroblasts. Embryos (n = 288) remained 26 to 32 hours in these culture systems. Video tape recording for later morphological analysis (11 parameters) was performed on 117 conventionally cultured and 104 cocultured embryos, shortly before replacement, by an independent observer, unaware of the culture conditions for each embryo. A significantly greater number of cocultured embryos (52%) had "good" morphology (zero or only one abnormal characteristic) as compared with conventionally cultured embryos (30%). The most outstanding morphological characteristic of cocultured embryos was the expanded appearance of their blastomeres. The incidence of implantation per embryo increased from 13% to 19% when the coculture rather than conventional culture system was used, and the incidence of ongoing pregnancy per patient after coculture doubled to 35%.

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

Human zygotes resulting from IVF were placed in two different culture systems to evaluate in-vitro development and to establish pregnancies in patients following embryo replacement. Treatment A (control) consisted of culturing zygotes in a modified Earle's Balanced Salt solution while treatment B consisted of culturing zygotes on a monolayer of fetal bovine uterine fibroblasts in this same culture medium. At the time of embryo replacement, embryos within treatments A and B had 3.7 and 4.3 blastomeres present, respectively. After 24 h in culture, the cellular fragmentation rate for treatment A embryos was 0.85 which was greater (P less than 0.05) than the fragmentation rate of 0.40 for embryos within treatment B. The incidence of implantation for patients whose embryos were given treatment A was 17.0% which was lower (P less than 0.05) than 35% for those given treatment B. Implantation rates increased with time in culture (43%) for treatment B embryos. Culture by treatment B of three-pronucleate zygotes resulted in 7/9 and 4/9 reaching the blastocyst and expanded blastocyst stages, respectively, whereas only 1/26 three-pronucleate zygotes cultured using treatment A reached either of these stages.

Prognostic value of morphologic characteristics of cryopreserved embryos: a study using videocinematography.

Fifty embryos, previously frozen at the zygote or 2- to 4-cell stage, were studied. Observers, who were unaware of the occurrence of pregnancy, assessed the morphologic features of these embryos by retrospectively analyzing videocinematographic recordings that were made shortly before replacement. Seven of 26 zygotes and 4 of 24 cleaved embryos implanted, with the incidence of implantation being analyzed in relation to 12 morphologic characteristics. Smooth surface of the blastomere's membranes was a statistically significant predictor of implantation for frozen-cleaved embryos, and variable zona pellucida thickness was the only parameter with predictive value for frozen zygotes. A highly significant difference was found between the implanting capacity of previously frozen-cleaved embryos and the number of abnormal morphologic characteristics. Three quarters of the thawed embryos had at least two abnormal characteristics, indicating that cryodamage was high. Major advantages of videocinematography are the absence of time constraints associated with observing live embryos, the ability to observe new morphologic parameters by freeze-frame and slow motion, and the permanent storage of embryonic data for quality control evaluation.

Semen characteristics, scrotal circumference and bacterial isolates of fine wool range rams.

During 1983, 887 fine wool rams were subjected to breeding soundness evaluation to determine effects of age and scrotal circumference on semen characteristics. Of rams evaluated, 94.4, 84.8, and 81.9% of young (6 yr), respectively, were rated as satis-factory. Old rams had fewer (P < 0.05) motile cells and more (P < 0.05) abnormal cells than young rams. Among older rams there was a higher percentage (P < 0.05) with testicular lesions than among young or mature rams. Scrotal circumference was positively correlated (P < 0.01) with semen volume and percentage of motile normal cells. Motility was negatively correlated (P < 0.01) with percentage of abnormal cells. Mature and old rams with large (>/=36.7 cm) testis had more (P < 0.10) abnormal cells than those in the same age groups with smaller testis. Scrotal circumference was positively correlated (P < 0.10) to volume and motility in mature and old rams, while motility was negatively correlated (P < 0.01) to percentage of abnormal cells in all age groups. Previous semen testing reduced (P < 0.05) the percentage of mature rams with leucocytes. Vaccination against epididymitis reduced (P < 0.05) the incidence of mature rams with leucocytes and testicular lesions. Brucella ovis was recovered from 54 (67.5%) of 80 ejaculates cultured. Among rams infected with B . ovis , only three (5.6%) were vaccinated against B . ovis and had their semen tested previously.