Kurt F. Miller

Follicle size and implantation of embryos from in vitro fertilization

Objective To determine the effect of increased follicle size at the time of hCG administration on the outcome of in vitro fertilization (IVF). Methods Fifty women immediately before and 50 immediately after a protocol change were reviewed retrospectively. The criterion for hCG administration was changed from a leading follicle size of at least 17 mm to one of at least 20 mm (average of two dimensions). Changes in follicle size and number, oocytes recovered, fertilization rate, embryo development, implantation rate, and pregnancy rate were examined. Women over 40 years of age, those undergoing intracytoplasmic sperm injection, and those whose stimulation did not include GnRH agonists were excluded. Results The number of large follicles increased, but peak estradiol concentrations and number of oocytes recovered did not differ when hCG was administered at a follicular size of at least 20 mm. The numbers of oocytes fertilized, embryos replaced, and embryos cryopreserved were not statistically different. The percentage of cleaved fertilized oocytes was greater and the degree of embryo fragmentation smaller when hCG was administered at an increased follicular size. The implantation rate was higher and clinical and ongoing pregnancy rates tended to be higher when hCG was administered at a larger follicle size. Conclusion The administration of hCG at a larger follicle size appears to have a beneficial effect on the outcome of IVF. Embryo quality—as reflected by decreased fragmentation, increased cleavage, and increased implantation rate—appears to be improved when hCG is delayed until two or more follicles reach at least 20 mm in diameter.

The significance of elevated progesterone at the time of administration of human chorionic gonadotropin may be related to luteal support

AbstractPurpose: Our purpose was to determine if the progesterone concentration on the day of HCG administration is associated with the establishment of pregnancy in IVF patients. Methods: Concentrations of progesterone on the day of hCG were examined retrospectively in 293 patients with ovarian hyperstimulation induced by GnRH agonist with hMG and/or FSH. Patients were grouped based on progesterone concentration: <0.9, 0.9 to <1.2, 1.2 to <1.5, and ≥1.5 ng/ml. Oocytes recovered, fertilization rate, and pregnancy rate were compared among groups. Results: Patients with a higher concentration of progesterone had increased concentrations of estradiol and greater numbers of oocytes retrieved. No differences were found for fertilization rate, polyploid fertilization, or pregnancy rate. It was noted that there was an association between the type of luteal support and the effect of high preovulatory progesterone on pregnancy rate. Conclusions: These data suggest that oocytes and embryos derived from patients with high preovulatory progesterone are not of a reduced quality. The association of high progesterone concentrations with a reduced rate of pregnancy varied with the type of luteal support.

Covering embryo cultures with mineral oil alters embryo growth by acting as a sink for an embryotoxic substance

PurposeAssisted reproductive technologies require the use of culture media for gametes and embryos. Culture media and conditions intended for use in human in vitro fertilization are tested prior to use by evaluating their ability to support normal development of mouse embryos.ResultsDevelopment to blastocysts of two-cell mouse embryos was equivalent for embryos cultured in center well dishes alone (93±10%, mean±SD) and embryos cultured in center well dishes with an overlay of 1 ml of mineral oil (94±5%). In contrast, no development of two-cell-stage mouse embryos occurred in an embryotoxic medium when cultured in center well dishes. However, when this medium was covered with an overlay of mineral oil, development to blastocyst was no different than that of controls (96±5%). Similar results were found with cultures of one-cell-stage mouse embryos.ConclusionThese results indicate that in some circumstances the presence of mineral oil can significantly alter the outcome of embryo culture. Further, these results reinforce the necessity of directly testing the exact culture conditions to be used for assisted reproductive technologies.

In vitro development and implantation rates of fresh and cryopreserved sibling zygotes

Objective To compare the developmental potential of cryopreserved human zygotes after thawing to sibling zygotes that were transferred without cryopreservation. Methods Retrospective analysis of embryo data and pregnancy outcome for all in vitro fertilization (IVF) patients who had sufficient zygotes to allow fresh embryo transfer and cryopreservation of additional sibling zygotes for later use. Results Zygotes survived cryopreservation at a high rate (87%). After thawing, cryopreserved zygotes developed at rates similar to those of fresh zygotes. Pregnancy occurred at similar rates after replacement of fresh embryos (27.9%) or replacement of cryopreserved-thawed zygote-derived embryos (24.3%). Conclusions Human embryos can be cryopreserved at the pronuclear zygote stage with little loss of developmental potential. Cryopreservation allowed a reduction in the number of embryos transferred during an IVF retrieval cycle, thereby reducing the occurrence of multiple pregnancy. The total cumulative pregnancy rate per retrieval cycle doubled when pregnancies from cryopreserved-thawed zygotes were added to those originating from fresh zygotes.

Previous fertilization failure with conventional in vitro fertilization is associated with poor outcome of intracytoplasmic sperm injection

OBJECTIVE To evaluate the outcome of intracytoplasmic sperm injection (ICSI) in patients with a history of fertilization failure with conventional IVF. DESIGN Retrospective analysis of 2 years of clinical experience with ICSI. SETTING Clinical IVF-ET program in a tertiary care referral center. PATIENT(S) The results of ICSI performed between January 1995 and December 1996 were compared between patients with normal semen parameters and a history of fertilization failure (< 20% of oocytes) with conventional IVF and patients with male factor infertility. INTERVENTION(S) In vitro fertilization with ICSI. MAIN OUTCOME MEASURE(S) Parameters examined included oocyte survival, fertilization, embryo cleavage, implantation rates, and clinical pregnancy rates. RESULT(S) Fertilization was achieved with ICSI for all patients during the study period. Although oocyte survival and fertilization did not differ between groups, the pregnancy and implantation rates for patients with a history of idiopathic fertilization failure (20% and 6%, respectively) were significantly lower than those for other patients undergoing ICSI (47% and 22%, respectively). CONCLUSION(S) The outcome of ICSI varied depending on the indication for treatment. Patients who had a history of failed or poor fertilization in vitro with apparently normal semen parameters had significantly lower pregnancy and implantation rates than did patients with either obstructive azoospermia or impaired semen quality.

ENDOMETRIOSIS : MEDICAL AND SURGICAL INTERVENTION

Pregnancy rates with endometriosis-associated infertility may be improved by laparoscopic surgery or laparotomy for moderate to severe disease. Surgery for minimal to mild disease does not increase pregnancy rates. Medical treatment has not been shown to increase fecundity for any stage of the disease. Pregnancy rates with assisted reproductive technology for endometriosis appear to be comparable with those for tubal disease that are also treated with assisted reproductive technology. Medical and surgical treatments for pelvic pain with endometriosis are both effective, but surgery avoids the side-effects associated with drugs and may result in a lower recurrence rate.

Effect of metformin on mouse embryo development.

During controlled clinical trials, plasma metformin levels do not exceed 5 g/mL, even at maximum doses (10). Metformin hydrochloride (N, N-dimethylimidodicarbonimidic diamide hydrochloride; Sigma, St. Louis, MO) was dissolved in human tubal fluid (HTF) media (Irvine Scientific, Santa Ana, CA) to give working concentrations of 5, 25, and 100 g. The culture dishes containing 1 mL of media at each concentration were incubated for overnight equilibration at 37°C and 5% CO2. Thawed two-cell mouse embryos (Embryotech, Wilmington, MA) were pooled and randomly distributed into the culture dishes containing the various concentrations of metformin. The control consisted of HTF media alone (group 1). Groups 2, 3, and 4 were supplemented with metformin (5, 25, and 100g/mL). There were 40 embryos in each group. The embryos were incubated in an atmosphere of 5% CO2 at 37°C for 72 hours. The blastocyst development rate (BDR) was then calculated by dividing the number of embryos reaching the blastocyst stage by the total number of embryos cultured. The relationship between BDR and concentration was assessed with repeated measures logistic regression using generalized estimated equation (GEE) methodology with a compound symmetry correlation structure. The sample size was sufficient to detect whether a specific concentration reduced the odds of development by a factor of 2.5. Statistical significance was assessed using two-tailed P.05. Statistical computations were performed with SAS version 8.1 (SAS Institute Inc, Cary, NC). The BDR for groups 1, 2, and 3 were 94.7%, 92.5%, and 91.8%, respectively. These were not significantly different. However, the BDR of 7.5% for group 4 was significantly lower than the group 1 (control) ( P.004; Fig. 1). The embryos in group 4 were arrested at early stages of development and contained extensive fragmentation. The introduction of metformin has helped avert the need for parenteral gonadotropin superovulation in many CC resistant PCOS patients with hyperinsulinemia. It significantly decreased the waist-to-hip ratio, serum testosterone, fasting free fatty acid and insulin concentrations, and improved oxidative glucose utilization and menstrual cyclicity (1–3). Despite numerous studies dealing with the hormonal, metabolic, morphometric, and clinical effects of metformin therapy in PCOS patients, little is known about possible effects on oocyte quality and early embryogenesis. The reported maximum serum concentration of metformin did not exceed 5g/mL with the maximum oral dose (10). We suspect that the early embryo would not be exposed to higher concentrations than that, though metformin levels in the follicular fluid are unknown. Normal embryonic development occurred at a concentration fivefold higher than the maximum clinical serum level. The toxic effects of metformin on in vitro development of mouse embryos only appeared at a concentration of 100 g/mL, which is never achieved in vivo.

The Effect of Platelet Activating Factor on Different Phases of Murine in Vitro Fertilization

PurposeOur purpose was to examine the effect of platelet activating factor (PAF) in different phases of mouse in vitro fertilization and optimal parameters that would enhance the fertilization rate.Design and SettingsVarious PAF concentrations (10−7 to 10−5 M) were selected to investigate its effect on three phases of mouse in vitro fertilization (i.e., sperm capacitation, sperm/oocyte coincubation, and preimplantation embryo growth) in three experimental groups: (I) with PAF treatment in the first phase, (II) with PAF treatment adopted in the first and second phases, and (III) with PAF treatment implemented in all three phases.ResultsThe improvement of the fertilization rate in PAF treatment groups over the control group ranges from 6.5 to 19.0% (P < 0.05 –P < 0.001). The highest enhancement of fertilization rate was achieved under the condition of PAF treatment (10−6 M) through sperm capacitation and sperm/oocyte coincubation phases.ConclusionThe PAF concentration of 10−6 M in sperm capacitation and sperm/oocyte coincubation yielded the greatest improvement in fertilization. However, continuing PAF treatment after sperm/oocyte coincubation had no beneficial effect on fertilization and preimplantation development.

Relationship between cytokines and the embryotoxicity of hydrosalpingeal fluid

Objective: The exact chemical composition of hydrosalpingeal fluid is unknown. The objective of this study was to characterize cytokines in hydrosalpingeal fluid (HSF) and examine their possible role in the embryo development.Study Design: HSF was aspirated at laparoscopic salpingectomy in eight infertile women. Levels of IL-1β, IL-13, IL-8, IL-6 and TNF-α in the HSF were determined by quantitative immunoassay kits. Two-cell mouse embryos were incubated with 0, 25, 50 and 75% concentrations of HSF. The blastocyst development rate (BDR) of mouse embryos was measured at each HSF concentration.Result(s): The embryotoxicity of HSF was concentration dependent. An increase in the HSF concentration resulted in significant decrease in % BDR (p < 0.01). IL-1β was present in six of the eight HSF samples with a mean (± SD) concentration of 0.9 ± 0.8 pg/mL. IL-13 was not detected in any of the HSF samples. IL-8, IL-6 and TNF-α were detected in all samples with a mean (± SD) concentration of 4741.2 ± 6554.4 pg/mL, 204.8± 132.8 pg/ml and 12 ± 12.8 pg/mL respectively. IL-6 was positively correlated with BDR (r = 0.53; p < 0.04).Conclusion(s): We demonstrated for the first time the presence of IL-1β, IL-8, IL-6 and TNF-α and the absence of IL-13 in human hydrosalpingeal fluid. IL-6 was positively related to the BDR.

Variation in recovery of motile sperm after preparation by a simple Percoll gradient technique

AbstractPurpose: Numerous techniques have been used to prepare sperm for assisted reproduction technology. Density-gradient centrifugation with Percoll is becoming a method of choice. This study reviewed the results of a simple two-layer discontinuous Percoll gradient. Methods: We reviewed retrospectively results obtained from 208 semen specimens prepared for in vitro fertilization by a discontinuous Percoll gradient. Results: Overall results (mean ± SD) were a recovery of 21±16% of total sperm, recovery of 38±30% of motile sperm, and a motility in the final sperm preparation of 88±10%. Specimens with higher initial concentrations had higher motilities in the final preparation. Higher total recovery and higher motility in the final preparation were found for specimens with a higher initial motility. Conclusions: This simple two-layer Percoll technique is rapid and inexpensive and reliably produces a final sperm preparation with desirable characteristics. Even though specimens with poor initial parameters yielded final preparations with excellent characteristics, fertilization rates were still related to the initial semen parameters.