Clinical application of intracytoplasmic sperm injection using in vitro cultured testicular spermatozoa obtained the day before egg retrieval.

OBJECTIVE: To study the effect of in vitro culture on the quality of human testicular sperm and the efficiency of intracytoplasmic sperm injection with in vitro cultured testicular sperm. DESIGN: Clinical study. SETTING: A private IVF center. PATIENT(S): Twenty consecutively seen IVF patients undergoing testicular biopsies for ICSI. INTERVENTION(S): The testicular specimens were cultured in vitro for 24 hours and the isolated spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Preincubation and postculture sperm motility, and fertilization, implantation, and pregnancy rates after intracytoplasmic sperm injection. RESULT(S): Motility increased from initial nonmotile or twitching sperm to free motile sperm in 18 of 20 cases. The injection of in vitro cultured testicular sperm resulted in a fertilization rate of 58%, an implantation rate of 20%, and a pregnancy rate of 45%. CONCLUSION(S): A testicular biopsy procedure can be performed the day before egg retrieval. Despite the low initial sperm quality, a high percentage of the prepared testicular sperm showed increased motility after 24 hours of culture. The injection of in vitro cultured testicular sperm into matured oocytes resulted in fertilization, implantation, and pregnancy rates comparable to those obtained with ejaculated sperm.

A comparison of post-thaw results between cryopreserved embryos derived from intracytoplasmic sperm injection and those from conventional IVF.

OBJECTIVE: To study the effect of freezing on early stage embryos derived from intracytoplasmic sperm injection (ICSI) or from IVF. DESIGN: Prospective, controlled clinical study. SETTING: Private IVF center. PATIENT(S): Sixty-seven consecutive patients undergoing frozen-thawed embryo transfer cycles. INTERVENTION(S): Early stage embryos were frozen, thawed, and transferred. MAIN OUTCOME MEASURE(S): Post-thaw survival, implantation and pregnancy rates. RESULT(S): We noted an 88% post-thaw survival rate, an 18% implantation rate, and a 52% pregnancy rate in the ICSI group and 81%, 11%, and 25%, respectively, with conventional fertilization. CONCLUSION(S): Early stage embryos (either zygote or 2-4 cells) derived from ICSI can be frozen with confidence and higher post-thaw survival and pregnancy rates can be achieved when compared with those from conventional IVF.

Maximizing pregnancy rates and limiting higher-order multiple conceptions by determining the optimal number of embryos to transfer based on quality.

OBJECTIVE: To define statistical thresholds for the number of embryos to be transferred to achieve an optimal pregnancy rate and keep higher-order multiple conceptions (pregnancy with more than two fetal sacs with cardiac activity) within an acceptable limit. DESIGN: A retrospective review of patient records. SETTING: Private practice assisted reproductive technology (ART) facility. PATIENT(S): Seven hundred fifty-four consecutive patients who underwent IVF-ET from 1994-1996. INTERVENTION(S): Embryo grading and score system used on day 3 of embryo transfer. MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and multiple conception rates. RESULT(S): For women < or =35 years old, transfer of up to four poor-quality, two fair-quality, or two good-quality embryos is optimal to eliminate any risk of higher-order multiple pregnancies. Transfer of four poor-quality, three fair-quality, or two good-quality embryos is recommended for women 36 to 39 years old. In women who are > or =40 years old, five embryos need to be transferred regardless of embryo quality. CONCLUSION(S): The mean cumulative embryo score can be used as a reference to determine an optimal number of embryos to transfer and to predict pregnancy outcome.

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.

Co-culture with assisted hatching of human embryos using Buffalo rat liver cells.

Commercially obtained Buffalo rat liver (BRL) cells were grown in monolayer culture. The effect of BRL cell co-culture with assisted hatching on embryo development, implantation and pregnancy was investigated in a population of 200 'first-time' in-vitro fertilization (IVF) patients, subdivided into three groups according to the methods of fertilization [IVF; intracytoplasmic sperm injection (ICSI); ICSI/IVF]. Assisted hatching was performed on all embryos chosen for transfer. Following co-culture, the overall embryo quality, implantation rate and pregnancy rates were not significantly different from the controls. However, when grouped according to fertilization method, co-culture was found to have an impact on pregnancy and implantation rates in the group undergoing conventional IVF. Using co-culture with assisted hatching, we were able to achieve a 58% (38/65) clinical pregnancy rate with a 49% (32/65) live birth rate and a 26% (60/235) implantation rate. No changes in the pregnancy and implantation rates were apparent in ICSI or ICSI/IVF subgroups. This is the first prospective, randomly controlled study which reports the use of BRL cell co-culture for human IVF for a large number of patients undergoing IVF for the first time.

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.

Characteristics of lidocaine block of sodium channels in single human atrial cells.

Although lidocaine block of cardiac Na+ current (INa) has been extensively studied in animal tissues, very little is known about its actions on human cardiac INa. We studied the effects of lidocaine (0.01-10 mM) on human atrial INa in single myocytes using whole-cell patch-clamp techniques. The dose-response relationship for lidocaine block at a low frequency (0.2 Hz, "tonic" block) indicated that lidocaine blocked Na+ channels by one-to-one binding with an apparent Kd of 291 microM. Lidocaine (200 microM) shifted the steady-state INa availability curve by -11 mV, but did not change the slope factor (n = 5). Lidocaine also induced use-dependent block that increased directly with increases in drug concentration (0.01-1 mM) and pulse duration (3-100 msec) and inversely with interpulse interval (2-0.33 sec). The time constant for onset of lidocaine (200 microM) block of INa displayed both a fast (tau f = 3.6 +/- 0.4 msec) and a slow (tau s = 168 +/- 21 msec) exponential component (n = 10). In addition, lidocaine slowed the rate of INa recovery after a 1-sec conditioning pulse to -20 mV, recovery was biexponential at a low drug concentration (20 microM), but had only a single slow phase at a high drug concentration (200 microM). These characteristics of lidocaine block suggest that lidocaine binds to both inactivated and activated Na+ channels in human atrial cells and that use-dependent block of INa by lidocaine is dependent on drug concentration, interpulse interval and pulse duration, findings similar to those reported for other mammalian species. The similarity of these results to those obtained from atrial as well as ventricular cells from other species suggests that some source other than differential drug action on atrial and ventricular INa underlies differential drug efficacy against supraventricular and ventricular dysrhythmias.

Amiodarone blocks the inward rectifier potassium channel in isolated guinea pig ventricular cells.

We examined the effects of amiodarone (5-20 microM) on both whole-cell inward rectifier potassium current (IK1) and single IK1 channel activity in isolated guinea pig ventricular myocytes using patch-clamp techniques. In whole-cell voltage-clamp experiments (n = 8), amiodarone (10-20 microM) caused only a small reduction of outward current at -50 mV (12 +/- 6%, no significant difference, N.S.). However, inward current was significantly reduced at -120 mV (21 +/- 7%; P < .05). When CdCl2 (100 microM) and tetrodotoxin (10 microM) were used to block inward Ca++ and Na+ current, respectively, amiodarone significantly reduced IK1 in both the inward (14 +/- 5% at -120 mV; P < .02) and outward (12 +/- 5% at -50 mV; P < .05; n = 11) directions. However, block required high drug concentrations (10-20 microM) and was slow in onset. In contrast, amiodarone did not affect membrane current when IK1 had been previously blocked by Ba++ (5 mM). In inside-out patch-clamp experiments, amiodarone (5 microM) reduced single IK1 channel open probability by increasing interburst interval (from 0.6 +/- 0.03 to 3.1 +/- 0.9 sec; n = 5; P < .05) with no significant difference in the duration of mean open and closed times or the number of shut events within a burst. The net result was that there was only a small change in both burst duration and single-channel kinetics within a burst. Complete channel block occurred after the increase in interburst interval (n = 6 of six cells).(ABSTRACT TRUNCATED AT 250 WORDS)