Robert Forman

High pregnancy rate after early human embryo freezing

Human embryos produced by in vitro fertilization (IVF) were frozen with 1,2-propanediol as a cryoprotectant. Embryo survival after thawing was related to the presence of a nucleus in frozen cells and decreased with the increasing number of cells in the frozen embryo. None of five embryos frozen 3 or 4 days after IVF survived when thawed. Of 48 early embryos (35 patients) frozen 1 or 2 days after IVF, 42 (87.5%) were transferred in 32 patients. Ten pregnancies were initiated after frozen embryo transfer (ET). If we exclude the three infertile patients who had sexual intercourse in the fertile period, the pregnancy rate for each patient who had 1- or 2-day frozen embryo(s) was 22% (7 of 32). One of the pregnancies was obtained after ET of a 1-cell pronucleated frozen and thawed embryo. The rate of ongoing pregnancies after triple fresh ET was 23%. In patients having four embryos obtained in a single IVF cycle, the expected overall liveborn rate in an IVF-ET program including embryo cryopreservation could theoretically equal that of natural human fertility.

Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation

Multiple follicular stimulation for IVF may be associated with greatly elevated serum E2 concentrations that are presumed to be antinidatory. This factor was analyzed in 825 consecutive embryo transfer cycles. The pregnancy rate decreased significantly after the transfer of one and two embryos in association with preovulatory E2 levels greater than the 90th percentile for the group (2320 pg/ml). The pregnancy rate did not vary with preovulatory E2 concentration following the transfer of three embryos. Highly significant correlations were noted between preovulatory E2 and early luteal phase concentrations of E2 and P. In a subgroup of 245 cycles, there were no significant relationships between implantation and early luteal phase levels of P or the ratio of E2/P. There was a small but nonsignificant tendency for the pregnancy rate to decrease in association with raised luteal E2. It is concluded that excessive E2 levels at the time of ovulation induction with hCG had an adverse effect on implantation when one or two embryos are transferred, but this may be overcome by the transfer of three embryos. The consequences for embryo transfer are discussed.

Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program

Certain factors influencing the success of embryo cryopreservation were analyzed from 124cycles of in vitro fertilization and embryo transfer (IVF-ET) program in which 193 1- or 2-day embryos were frozen and had already been thawed. There were 100 transfers of one or two surviving embryos from which 26 pregnancies were initiated. Several factors significantly influenced embryo survival after thawing. They were: (1) the developmental stage of frozen embryos; (2) the appearance of the embryo at the time of freezing; and (3) the mode of ovarian stimulation in the IVF cycle. The pregnancy rate after frozen-thawed embryo transfer was higher with 4-cell frozen embryos than with embryos at all other stages combined. There were also tendencies for the pregnancy rate to be higher if a spontaneous luteinizing hormone surge occurred in the transfer cycle or if the duration of embryo storage did not exceed 1 to 2months. The results obtained support a new policy in IVF-ET programs: it should be advantageous for the sterile couple if the immediate fresh embryo transfer is only performed with the categories of embryos that demonstrate a poor aptitude for survival following cryopreservation procedures.

A new approach to follicular stimulation for in vitro fertilization: programed oocyte retrieval

Programed oocyte retrieval was performed in a group of 35 patients undergoing in vitro fertilization (IVF) treatment. The date of follicular aspiration was decided several months in advance and the cycle prior to oocyte recovery was modified with a progestagen or an estrogen-progestagen contraceptive pill. This was followed by a fixed-schedule ovulation stimulation and induction regimen. Follicular growth was not monitored. Thirty-four of the 35 patients had follicular aspiration, and at least one embryo was obtained in 30 of them. The clinical pregnancy rate (excluding cryopreserved embryos) was 20% per IVF cycle, 21% per attempted oocyte retrieval procedure, and 23% per embryo transfer cycle. Programed oocyte retrieval is a realistic option for follicular stimulation for IVF treatment and is associated with significant practical and economic benefits.

Comparison between flare up and down regulation effects of luteinizing hormone-releasing hormone agonists in an in vitro fertilization program

Luteinizing hormone-releasing hormone (LH-RH) agonists are being increasingly used in ovulation stimulation protocols in IVF programs. The results of two methods of utilization of LH-RH agonists are compared. In the long protocol, gonadotropin stimulation was commenced only after a preliminary period of pituitary desensitization with LH-RH agonist. In the short protocol, exogenous gonadotropins were administered shortly after the start of LH-RH agonist therapy, benefiting from the gonadotropin flare up effect. One hundred eighty-six patients were equally divided between the two treatments. There was no difference in the ovarian response on the day of human chorionic gonadotropin (hCG) or the number of mature oocytes recovered. The cleavage rate of mature oocytes was higher in the short protocol (70% versus 56% P less than 0.01). The ongoing pregnancy rate per treatment cycle was similar in both groups (18% in the long protocol and 16% in the short protocol). Analysis of the luteal phases revealed a trend for higher progesterone values in the long protocol although this was only significant on the 2nd day following oocyte retrieval. As the clinical results were similar, other factors should be taken into account when deciding therapy. These include patient convenience, cost, and side effects. Other schedules of ovulation stimulation with LH-RH agonists are discussed.

Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology

The corpus luteum-endometrial unit was investigated in in vitro fertilization (IVF) cycles using endocrine, morphologic, and biochemical measurements on the day normally scheduled for embryo transfer (day 16), in 12 stimulated and 4 natural cycles. Advanced endometrial histologic maturity was recorded in 9 of the 12 stimulated cycles. No in-phase endometria were seen when the preovulatory plasma estradiol (E2) was greater than 500 pg/ml or the day 16 plasma progesterone (P) greater than 10 ng/ml in natural or stimulated cycles. Significant negative correlations were noted between both preovulatory E2 and day 16 P and the concentration of cytosolic progesterone receptor (PRc). Advanced endometrial maturity tended to be associated with low concentrations of PRc. Regardless of endometrial maturity, the natural cycle was characterized by low cytosolic E2 receptors (ERc) and high PRc, whereas the concentration of both receptors was usually greatly reduced in stimulated cycles. It is concluded that the advanced endometrial maturation observed in stimulated IVF cycles is a consequence of the production of supraphysiologic levels of sex steroids by the corpus luteum that cause profound modifications of endometrial receptor dynamics.

Human embryo viability related to freezing and thawing procedures

Freezing and thawing procedures did not affect the chances of successful transfer of human embryos. The postimplantation viability of frozen-thawed embryos was equivalent to that of fresh, immediately transferred embryos. Selection of the embryos that were more suitable for freezing did influence the pregnancy rate after transfer of the remaining fresh embryos. The therapeutic efficacy of in vitro fertilization and embryo transfer programs will be greatly increased if only one embryo is transferred in the in vitro fertilization cycle, the others being separately frozen for further embryo transfer in subsequent cycles.

Systematic semen culture and its influence on IVF management

Semen specimens from 519 men were cultured for organisms 2 weeks before oocyte recovery for in‐vitro fertilization (IVF). The cultures from 72 men contained more than 5000 bacteria/ml and the IVF attempt was postponed. The IVF outcome was assessed in 382 couples in relation to the semen culture result. The pregnancy rate per cycle was significantly reduced when the semen culture contained organisms compared with axenic semen (P<0‐05). This was independent of the cleavage rate of oocytes and the number of embryos transferred. Although mycoplasma was frequently cultured, chlamydial infection was very rare and no viruses were isolated in this study.

Importance of semen preparation in avoidance of reduced in vitro fertilization results attributable to bacteria

Although the physical treatment of semen for IVF and related procedures is sufficient to remove most organisms present in semen, the pathogenic varieties tend to be more resistant. The supplementation of oocyte culture medium with both penicillin and streptomycin was associated with the eradication of 100% of organisms in the current study. In these circumstances, the presence of pathogenic organisms in the untreated semen is not associated with reduced oocyte fertilization in vitro.

Improvements in Ovarian Stimulation for in Vitro Fertilization

What are the major improvements in stimulation over recent years? The stimulated cycle has completely superseded the natural cycle since this is the only method of obtaining several embryos. In conventional stimulation, despite an enormous body of literature, no single treatment modality has emerged as superior to other forms of therapy. Undoubtedly it is the experience and habits of individual IVF teams that determine the success of one or another method. Therefore programmed stimulation is an improvement in that it introduces the factors of convenience and simplicity while retaining the clinical efficacy of stimulation by conventional treatment. The analogues of LHRH are still in an early phase of development, but they appear to be associated with a low cancellation rate even in previous poor-responders. Furthermore, preliminary results seem to indicate that this is the first development since the widespread adoption of follicular stimulation, which is associated with an increased pregnancy rate. We are currently involved in trying to associate programmed stimulation and analogue treatment in a bid to combine the advantages of both therapies. Finally, embryo cryopreservation must be mentioned. There is no point in refining our stimulation protocols to obtain many embryos if those surplus to immediate requirement are disposed of. Cryopreservation offers the means of maximizing the pregnancy potential of a single IVF cycle while reducing the possibility of multiple pregnancy.