André Hazout

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.

ANTIOXIDANTS TO REDUCE SPERM DNA FRAGMENTATION: AN UNEXPECTED ADVERSE EFFECT

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (-19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

High-magnification ICSI overcomes paternal effect resistant to conventional ICSI

Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.

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, Hôpital 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.

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.

Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles.

OBJECTIVE To present our experience using cocultured cryopreserved and transferred blastocysts. DESIGN Retrospective study of patients undergoing transfer of cryopreserved blastocysts. SETTING Three different IVF centers. PATIENTS Four hundred sixty-seven thawed cycles from January 1991 to June 1994. MAIN OUTCOME MEASURE Pregnancy rate per cycle after transfer of pre-embryos developed from thawed blastocysts. RESULTS One thousand two hundred thirty-nine blastocysts were thawed. Of these, 1,033 (83%) survived thawing and were transferred. Five hundred sixty-three thawed cycles resulted in 516 (92%) receiving intrauterine transfer. One hundred twelve clinical pregnancies were established, resulting in a 21.7% pregnancy per transfer with a 19% ongoing rate. The implantation rate of 13.4% results from 138 implanted pre-embryos. There was a higher PR in the programmed cycle (79/302; 26.2%) compared with the natural cycle (6/47;13%). CONCLUSIONS Freezing at the blastocyst stage is a proven and reliable method in IVF technology. Although there may be fewer pre-embryos, their ability to implant appears to approach the potential of a fresh transfer.

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.

Luteinizing hormone affects uterine receptivity independently of ovarian function

Previous studies have suggested that LH, in addition to its well-known effects on the ovary, may exert direct effects on the uterus. This study evaluated the effects of mid-cycle administration of human chorionic gonadotrophin (HCG), which signals through the LH receptor, on endometrial thickness and uterine receptivity in two groups of women lacking ovarian activity and receiving embryos from an oocyte donation programme. Patients in one group still had ovulatory cycles, but their ovarian function was suppressed by pituitary down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist in the embryo transfer cycle, resulting in low endogenous LH concentrations. Patients in the other group were menopausal women whose pituitary function was not down-regulated in the embryo transfer cycle and whose endogenous LH concentrations were thus high. Patients in each of the two groups were randomized into two subgroups. Patients in one subgroup were given 5000 IU of HCG 2 days before oocyte recovery in the corresponding donor. Patients in the other subgroup received placebo at the same time. Oocytes from each donor were randomly distributed between one patient from the HCG subgroup and one patient from the placebo subgroup in each patient group. Endometrial growth and secretory transformation were stimulated by sequential treatment with oestradiol valerate and progesterone. In women with low endogenous LH receiving placebo, endometrial thickness stopped increasing at the beginning of secretory transformation. Mid-cycle HCG administration resulted in a continuous increase in endometrial thickness through this period, improved the implantation rate after embryo transfer in these women (30.6 versus 20.7%) and augmented the number of multiple pregnancies. No similar stagnation of endometrial thickness and no effects of mid-cycle HCG administration on endometrial thickness, the implantation rate and the number of multiple pregnancies were found in women with high endogenous LH. It is concluded that endometrial maturation is disturbed in women with low endogenous LH but can be rescued by mid-cycle stimulation of LH receptor with exogenous HCG in the absence of ovarian activity.

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.

Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination

More than 17,000 intrauterine insemination (lUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.