Premature elevation of plasma progesterone alters pregnancy rates of in vitro fertilization and embryo transfer

OBJECTIVE: To determine if an increase in plasma P occurring before hCG administration might impair the outcome of IVF-ET. DESIGN: Five hundred eighty-five IVF-ET cycles were prospectively studied for the occurrence of plasma P elevation before hCG administration. SETTING: Tertiary institution, IVF-ET program, H?pital A. Béclère. PATIENTS: Participating patients included IVF-ET candidates 23 to 42 years of age only, excluding the couples in whom a male factor was a primary or an accessory cause of infertility. MAIN OUTCOME MEASURES: To clarify the practical consequences on IVF-ET outcome of pre-hCG increases in plasma P, we studied 585 consecutive IVF-ET cycles. These were divided into two groups according to plasma P levels observed on the day of hCG administration; plasma P of 0.9 ng/mL (2.9 nmol/L) was taken as an arbitrary cutoff value. Group A included 485 IVF cycles in which plasma P was < or = 0.9 ng/mL (2.9 nmol/L); group B included the remaining 100 cycles in which plasma P was > 0.9 ng/mL (2.9 nmol/L). RESULTS: The number of mature oocytes retrieved, the oocyte cleavage rate, and the number of embryos obtained were similar in groups A and B. In contrast to this apparent similarity in oocyte quality, a decrease in pregnancy rate (PR) and a trend for a decrease in embryo implantation rate were observed in group B in comparison with group A. CONCLUSIONS: The similar fertilization and cleavage rates obtained in groups A and B suggest that pre-hCG elevation in plasma P does not lead to decreased oocyte quality. Yet the lower PR observed when plasma P rises prematurely suggests that the prolonged but discrete elevation in plasma P occurring in these cases might alter endometrium receptivity to embryo implantation.     

Computerized assessment of endometrial echogenicity: clues to the endometrial effects of premature progesterone elevation

OBJECTIVE: To determine whether premature progesterone elevation affects the timing of hyperechogenic transformation of the endometrium during the early luteal phase of controlled ovarian hyperstimulation (COH) cycles. DESIGN: Prospective analysis. SETTING: Assisted Reproduction Unit, H?pital Antoine Béclère, Clamart, France. PATIENT(S): Fifty-nine women undergoing 59 IVF-ET cycles. INTERVENTION(S): Patients underwent COH with a GnRH agonist and hMG. Endometrial echogenicity was assessed on the days of hCG administration, oocyte retrieval, and ET. Results are expressed as the extent of submyometrial hyperechogenic area in relation to the total endometrial surface as determined by a computer-assisted analysis system. Patients were sorted according to whether their plasma progesterone level exceeded 0.9 ng/mL (n = 26) or not (n = 33) on the day of hCG administration. MAIN OUTCOME MEASURE(S): Endometrial echogenicity. RESULT(S): On the day of hCG administration, the degree of endometrial echogenicity was similar in both groups (41% vs. 40%), but after hCG administration, it increased significantly faster in the high progesterone group than in the low progesterone group (70% vs. 63% at oocyte retrieval and 90% vs. 79% at ET, respectively). CONCLUSION(S): End-follicular phase elevation in plasma progesterone (>0.9 ng/mL on the day of hCG administration) was associated with a faster increase in endometrial echogenicity during the early luteal phase of COH cycles. This observation is consistent with the hypothesis that premature progesterone elevation hastens the secretory transformation of the endometrium.     

Induction of subcutaneous tissue fibrosis in newborn mice by transforming growth factor beta--simultaneous application with basic fibroblast growth factor causes persistent fibrosis

OBJECTIVE: To determine the effect of serum P on endometrial histology in stimulated cycles. DESIGN: Prospective clinical study. SETTING: Community hospital-based donor oocyte program. PATIENT(S): Fertile young oocyte donors and infertile donor oocyte recipients. INTERVENTION(S): Oocyte donors underwent gonadotropin stimulation after midluteal pituitary suppression. Endometrial biopsies were obtained at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Endometrial histology and serum P levels in oocyte donors. Pregnancy and implantation rates in oocyte recipients. RESULT(S): Thirteen biopsy specimens (52.0%) showed in-phase mixed proliferative pattern (days 14 to 15), whereas 12 (48.0%) were secretory (days 16 to 17). On the day of hCG, subjects with secretory endometrium had higher P of 1.7 ng/mL (5.4 nmol/L) than women with the mixed pattern (0.8 ng/mL [2.5 nmol/L]). Progesterone > or = 0.9 ng/mL had a 78.6% positive predictive value for secretory transformation. In 75.0% of cycles with secretory endometrium, P was > or = 0.9 ng/mL, (2.9 nmol/L) as early as 2 days before hCG. Both mixed and secretory patterns were associated with similar clinical pregnancy rates (57.1% and 60.0%, respectively) and delivery rates (38.1% and 50.0%, respectively) in recipients. CONCLUSION(S): Subtle elevation of P induced secretory endometrial transformation without reduction in embryo viability.     

Elevation of the serum bilirubin diconjugate fraction provides an early marker for cholestasis in the rat

OBJECTIVES: To determine whether the sisters of women with premature ovarian failure (POF) showed a response to gonadotropin stimulation comparable to that of anonymous ovum donors. DESIGN: Historical cohort study. SETTING: Records of 228 consecutive ovum recipients in an academic assisted reproductive technology program. PATIENT(S): Criteria for inclusion were oocyte recipients age < or = 40 years, FSH > 18 mIU/mL (conversion factor to SI unit, 1.00), and/or failure to respond appropriately to controlled ovarian hyperstimulation (COH). Seventy-nine recipients were classified on the basis of whether they received oocytes from anonymous donors (group I, n = 66) or sister donors (group II, n = 13). MAIN OUTCOME MEASURE(S): Controlled ovarian hyperstimulation response, pregnancy rates (PRs), and implantation rates. RESULT(S): The ages of the donors to groups I and II were comparable (31.1 +/- 16.7 versus 29.8 +/- 7.2 years), but those in group II exhibited a higher baseline FSH level (12.8 +/- 2.1 versus 8.6 +/- 5.8 mIU/mL). Group II versus I had a relative risk of 5.1 for cancellation (4 of 13 [30.8%] versus 4 of 66 [6.1%], respectively). In completed cycles of groups I and II, respectively, there was no difference in serum E2 on the day of hCG administration (2,356 +/- 826 versus 1,847 +/- 843 pg/mL; conversion factor to SI unit, 3,671), number of oocytes retrieved (25 +/- 14 versus 22 +/- 13), number of embryos transferred (4.4 +/- 2.1 versus 4.0 +/- 1.0), spontaneous abortion rate (22.7% versus 25.0%), PR (35.5% versus 36.4%), and implantation rate (16.2% versus 16.4%). CONCLUSION(S): There is an increased cancellation rate and, consequently, an overall trend toward decreased ovarian response to gonadotropin stimulation in the sisters of patients with POF. Despite these factors, the implantation rates and PRs of embryos derived from patients reaching retrieval were similar to those from anonymous donors. We recommend counseling women with POF that their sisters may not be ideal ovum donors.     

Serotonin 5-HT4 receptor agonistic activity of the optical isomers of (+/-)-4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro-2,3- dihydro-2-methylbenzo[b]furan-7-carboxamide

OBJECTIVE: To compare the influence of incongruent (asymmetric) follicular development on treatment outcome in IVF-ET and GIFT cycles. DESIGN: A retrospective comparative study. SETTING: Tertiary referral center for infertility. PATIENT(S): Five hundred forty-three consecutive assisted reproduction cycles (428 IVF-ET and 115 GIFT) in 422 infertile patients. INTERVENTION(S): Controlled ovarian hyperstimulation (COH) and IVF-ET or GIFT. MAIN OUTCOME MEASURE(S): The incongruity ratio as a parameter of the asymmetry in follicular development and pregnancy rate (PR). RESULT(S): For GIFT cycles, the PRs were 37.8% and 15.7% in cycles with congruent and incongruent follicular development, respectively. However, for IVF-ET cycles, the PR was not affected by incongruent follicular development: 28.2% and 29.0%, respectively. An inverse relationship was observed between the degree of incongruity and the estimated probability of pregnancy in GIFT cycles but not in IVF-ET cycles. Neither the side of the dominant ovary nor the degree of incongruity were consistent in consecutive cycles. CONCLUSION(S): Incongruent follicular development during COH has a significantly negative influence on the outcome of GIFT cycles but not on the outcome of IVF-ET cycles. The reason for this difference is not clear. We recommend considering IVF-ET instead of GIFT if incongruent follicular development occurs.     

The predictive value of uterine artery blood flow measurements for uterine receptivity in an intracytoplasmic sperm injection program

OBJECTIVE: To assess whether uterine artery blood flow impedance, measured as the pulsatility index on the day of ET in patients undergoing IVF-ET with microinjection, can predict the likelihood of pregnancy. DESIGN: Prospective clinical study. SETTING: A tertiary referral center for assisted reproduction. PATIENT(S): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) for andrologic indications. INTERVENTION(S): Transvaginal color Doppler examination performed on the day of ET. MAIN OUTCOME MEASURE(S): Mean (+/- SD) pulsatility index value of the left and right uterine arteries, serum E2 levels, implantation rates, and ongoing pregnancy rates (PRs). RESULT(S): The patients were divided into pregnant and nonpregnant groups and were separated according to whether the pulsatility index was low (1.00-1.99), medium (2.00-2.99), or high (> or = 3.00). The pulsatility index values did not change statistically in the pregnant and nonpregnant groups. The implantation rates were 19.5%, 15.4%, and 25% for the low-, medium-, and high-pulsatility index groups, respectively. The ongoing PRs for the same groups were 35.3%, 26.7%, and 37.5%, respectively. CONCLUSION(S): The study suggests that blood flow, measured as the pulsatility index on the day of ET, cannot predict the likelihood of pregnancy in stimulated cycles of ICSI.     

Uterine contractions at the time of embryo transfer: a hindrance to implantation?

OBJECTIVES: To investigate the hormonal control and the possible consequences of uterine contractions (UC) on IVF-ET outcome. MATERIALS AND METHODS: We studied prospectively 220 controlled ovarian hyperstimulation (COH) cycles for IVF-ET. Just before ET, women underwent 5-minute digital recordings of the uterus using US image analysis software for UC assessment. Plasma progesterone (P) and estradiol were measured. Four groups were defined according to UC frequency: < or = 3.0 (n = 53), 3.1 to 4.0 (n = 50), 4.1 to 5.0 (n = 43), and > 5.0 (n = 74) UC/minute, respectively. RESULTS: Patients, COH and embryology characteristics were comparable in all groups. Notwithstanding estradiol levels were not associated with UC characteristics, plasma P and UC frequency were negatively correlated (r = -0.34, P < 0.001). A stepwise decrease in clinical and ongoing pregnancy as well as implantation rates occurred from the lowest to the highest UC frequency groups (53%, 36%, 21%; 46%, 32%, 20%; 23%, 19%, 10%; and 14%, 11%, 4%; P < 0.001). Direction of UC did not affect ET outcome. CONCLUSIONS: The negative correlation between UC frequency and P levels supports the utero-relaxing properties of P. High frequency UC on the day of ET hinder IVF-ET outcome, possibly by expelling embryos out of the uterine cavity.     

Natural cycle in vitro fertilization-embryo transfer at the University of Ottawa: an inefficient therapy for tubal infertility

OBJECTIVE: To describe our experience with natural cycle IVF making clinical and endocrine comparisons with our standard stimulated cycle IVF program. DESIGN: We attempted 75 natural IVF-ET cycles with hCG given to preempt the LH surge and compared these with 450 attempts at standard superovulation IVF-ET done in our unit during the same time period. PATIENTS: Natural cycle patients are normally ovulating women < age 38. Superovulation IVF-ET patients are all < 41 years old. Patients in both groups had partners with normal semen parameters and tubal factor infertility. MAIN OUTCOME MEASURES: Cancellation rates, pregnancy rates per egg retrieval, per ET procedure, and luteal phase E2:P ratios of the treatment cycles are compared. RESULTS: There were 35 of 75 (47%) natural cycle and 112 of 450 (25%) superovulation cycle cancellations. An egg was retrieved in only 24 of 40 (60%) natural cycles and 336 of 338 (99%) superovulation egg retrieval procedures. Pregnancy rates per ovum pick-up procedure were significantly higher: 65 of 338 (19%) in the superovulation versus 2 of 40 (5%) in the natural cycle groups. Pregnancy rates per ET were not significantly different between natural IVF-ET, 2 of 18 (11%) and superovulation IVF-ET, 65 of 298 (22%). The E2:P ratios 5 days after ET were similar in both groups at 18 +/- 4 after natural IVF-ET and 21 +/- 18 after superovulation IVF-ET. CONCLUSIONS: [1] Cancellation rates for natural cycle IVF are very high. [2] Midluteal E2:P ratios are the same in both groups. [3] Pregnancy rates per egg retrieval are significantly lower for natural versus superovulation IVF-ET. [4] In our experience, natural cycle IVF-ET is an inefficient therapy for tubal infertility compared with superovulation IVF-ET.     

Parvalbumin, a horizontal cell-associated calcium-binding protein in retinoblastoma eyes

OBJECTIVE: To evaluate the effect of prednisolone plus low-dose aspirin (PSL/LDA) in women with autoimmune conditions who were enrolled in an IVF-ET program. DESIGN: A retrospective clinical study. SETTING: In vitro fertilization unit, Niigata University Hospital, Niigata, Japan. PATIENT(S): Three hundred seven women who underwent IVF-ET between January 1996 and December 1997. INTERVENTION(S): Prednisolone (10 mg/d) and aspirin (81 mg/d) were administered to the women with autoantibodies who chose to participate. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rates with IVF-ET. RESULT(S): Women undergoing IVF who had positive antinuclear antibodies, with or without antiphospholipid antibodies, had significantly lower pregnancy and implantation rates than did women without autoantibodies (14.8% versus 21.7% and 6.8% versus 10.4%, respectively). The administration of PSL/LDA to women with antinuclear antibodies significantly improved the outcome of IVF-ET (40.6% pregnancy rate and 20.3% implantation rate). CONCLUSION(S): A high proportion of women who are undergoing IVF-ET have autoantibodies, which are associated with poor IVF outcomes. The administration of PSL/LDA to these women may improve their implantation rate.     

Culturing human embryos with and without glucose

OBJECTIVE: To review published data and to compare pregnancy rates (PRs) after culturing human embryos with and without glucose and phosphate. DESIGN: Comparison of results from various programs. SETTING: Assisted Reproductive Technology Program. PATIENT(S): Patients were enrolled in various studies. INTERVENTION(S): Human embryos were cultured with and without glucose and phosphate. MAIN OUTCOME MEASURE(S): Pregnancy rates after different techniques of embryo culture. RESULT(S): Some studies reported higher PRs in patients undergoing IVF after embryos were cultured in media without glucose and phosphate versus media with glucose and phosphate. One study showed that PRs were lower when embryos were cultured in media lacking glucose and phosphate compared with media containing glucose and phosphate. Some studies have also shown similar PRs with the two types of culture media. CONCLUSION(S): The PRs in IVF patients will not necessarily be enhanced if the embryos are cultured in media without glucose and phosphate.     

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.     

The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes

OBJECTIVE: To assess the effect of propofol on fertilization and early embryo development in a mouse IVF model. DESIGN: Controlled study. SETTING: Mouse IVF. INTERVENTION(S): Mouse oocytes were exposed in vitro to propofol at a concentration of 0 (control), 50, 250, 500, 1,000, or 5,000 ng/mL for 30 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the blastocyst stage was monitored daily. The potential to activate parthenogenetically oocytes also was evaluated by looking for spontaneous extrusion of the second polar body or development to the two-cell stage. In a second step, a pure propofol solution was added to culture medium and used as a standard. MAIN OUTCOME MEASURE(S): Two-cell and blastocyst-stage embryo. RESULT(S): Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of propofol solution in the medium, (i.e., 3% to 41%) in comparison with the control group (76%). Exposure of unfertilized oocytes for 30 minutes to propofol results in a parthenogenetic activation of 33% to 60%, which was significantly higher than the control (10%). When oocytes were kept in propofol for 24 hours, a mean of 30% of activation was observed as compared with 0.5% for the control. CONCLUSION(S): We can conclude from these experiments that even a brief exposure of cumulus-enclosed oocytes to a low concentration of propofol is deleterious to subsequent cleavage. Exposure of unfertilized oocytes to propofol results in a high degree of parthenogenetic activation.     

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.     

Physiopathology of premature progesterone elevation

OBJECTIVE: To determine the effects of the last hMG administration on plasma P and androgen profiles during controlled ovarian hyperstimulation (COH) for IVF-ET. DESIGN: Controlled clinical study. SETTING: The IVF-ET program of a tertiary outpatient care center, H?pital A. Béclère, Clamart, France. PATIENTS: Nine IVF-ET candidates aged 25 to 36 years having presented normal responses to COH in previous IVF-ET cycles. INTERVENTIONS: Controlled ovarian hyperstimulation was induced for IVF-ET using hMG after endogenous gonadotropins were suppressed with a time-release GnRH agonist. Just before the last hMG administration (225 IU), the participants were hospitalized for 24 hours for serial blood sampling. These occurred before (baseline) and after hMG administration, every 30 minutes for 1 hour, hourly for 4 hours, and every 3 hours for the remaining part of a 24-hour post-hMG observation period. MAIN OUTCOME MEASURE: Measurement of P, T, androstenedione (A), E2, FSH, and LH. RESULTS: Plasma P and androgens (T and A) increased significantly, reaching peak values 12 to 15 hours after hMG administration and decreased progressively thereafter, to reach values not significantly different from baseline 24 hours after hMG administration. Plasma E2 levels increased progressively and steadily during the 24-hour observation period. Plasma FSH levels remained constant after hMG administration while LH stayed undetectable. CONCLUSION: In COH cycles induced for IVF-ET, the hormonal profile after the last hMG injection suggests that hMG triggers an increase in plasma P and androgens that culminates 12 to 15 hours after hMG administration. This elevation in plasma P and androgens observed after hMG administration is likely to reflect a direct action of the LH and/or FSH components of hMG on granulosa cells. In some women these hormonal consequences of hMG treatment may impair endometrial receptivity in IVF-ET cycles.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.     

Changes in ribosomal ribonucleic acid content within in vitro-produced bovine embryos

The abundance of 28S, 18S, and 5S rRNA was measured by Northern blot techniques applied to RNA samples extracted from bovine oocytes and preattachment embryos produced by in vitro procedures. Total RNA content was estimated by comparing the intensity of hybridization signals of 28S and 18S rRNA probes to embryo RNA samples and to standard curves generated from bovine ovary or bovine oviduct cell RNA. RNA content declined from the oocyte to the morula stage (2.4 +/- 0.3 ng/oocyte, 1.7 +/- 0.5 ng/1-cell embryo, 2.2 +/- 0.9 ng/2- to 4-cell embryo, 0.8 +/- 0.2 ng/6- to 8-cell embryo, and 0.7 +/- 0.2 ng/morula). A marked increase in RNA content, based on levels of hybridization to 28S and 18S rRNA, was observed in blastocysts, in which values averaged 5.3 +/- 0.6 ng/embryo. On a relative basis, 5S rRNA abundance followed a pattern similar to that of 28S and 18S rRNA across the early development period to the blastocyst stage.     

Transfer of cryopreserved embryos improved pregnancy rates in patients with damage to the functional integrity of the sperm membrane as measured by the hypo-osmotic swelling test

OBJECTIVE: To compare the pregnancy rates (PRs) after transfer of cryopreserved embryos in patients who have damage to the functional integrity of the sperm membrane as measured by the hypo-osmotic swelling test to those without this defect. DESIGN: Prospective clinical study. SETTING: University-associated IVF center. PATIENTS: Fifty-four patients enrolled in a matched prospective study to evaluate the effects of low HOS scores (<50%) on PRs after IVF-ET were followed to determine the PR after transfer of cryopreserved embryos. MAIN OUTCOME MEASURE: Clinical PRs and implantation rates. RESULTS: Fourteen patients with low hypo-osmotic swelling test scores underwent 21 frozen ET cycles, achieved for clinical pregnancies for a PR per cycle of 19.0% and an implantation rate of 7.1%. Twelve patients with normal hypo-osmotic swelling test scores underwent 21 frozen ET cycles, achieved five preganancies for a clinical PR per cycle of 23.8% and an implantation rate of 9.3%. CONCLUSION: Previous studies have demonstrated an adverse effect of low hypo-osmotic swelling test scores on PRs after IVF-ET despite normal fertilization. This adverse effect was not found in the transfer of cryopreserved embryos from males with hypo-osmotic swelling test scores. Further investigation is required to determine how cryopreservation improves the chances of implantation of these embryos.     

Coculture of human embryos with buffalo rat liver cells for women with decreased prognosis in in vitro fertilization

OBJECTIVE: The coculture of human embryos with epithelial cells may improve both embryo quality and pregnancy rates. In this current study we tested the efficacy of coculture with the buffalo rat liver cell line on pregnancy rates in women with a potentially poor prognosis for success with in vitro fertilization (previous in vitro fertilization failure, advanced maternal age, increased early follicular follicle-stimulating hormone levels, and anovulation). STUDY DESIGN: This prospective controlled study evaluated a total of 203 women (135 coculture, 68 controls) undergoing in vitro fertilization. Implantation rates per embryo, clinical pregnancy rates, and continuing/delivered pregnancy rates were analyzed. RESULTS: Buffalo rat liver cells, which are commercially available, are stable in coculture. Implantation rates (number of sacs with fetal heart motion per embryos transferred) were similar for coculture (19%) and control (18%) embryos. No difference in the rate of continuing/delivered pregnancies per retrieval was noted (17% coculture vs 14% control) in the group with advanced maternal age, but coculture caused a trend toward improved pregnancy rates in the group with ovulatory dysfunction (43% coculture vs 14% control) and the group with previous in vitro fertilization failure (34% coculture vs 28% control). CONCLUSION: This is the first published controlled study to our knowledge that reports the use of the buffalo rat liver cell coculture for human in vitro fertilization in a large number of patients. Our data support consideration of buffalo rat liver coculture for in vitro fertilization for women with previous in vitro fertilization failure and possibly for patients with oocyte or ovulatory dysfunction.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.