Per Sundström

The success rate of in vitro fertilization of human oocytes in relation to the concentrations of different hormones in follicular fluid and peripheral plasma

Seventeen infertile patients were stimulated with clomiphene citrate and human chorionic gonadotrophin (hCG). During laparotomy or laparoscopy, follicles were aspirated. At least one fertilizable oocyte was obtained from 13 patients, who had normal preovulatory plasma 17 beta-estradiol (mean 2.2 nmol/l) in contrast to the 4 patients with infertilizable oocytes (mean 0.4 nmol/l). A close association was found between the success rate of fertilization and the increment of plasma progesterone from the day before to the day after the operation. If the concentrations of 17 beta-estradiol and progesterone in follicular fluid were below an arbitrary limit (17 beta-estradiol less than 500 nmol/l in association with progesterone less than 2,000 nmol/l), no oocytes could be fertilized. Apparently aspiration of oocytes and follicular fluid did not disturb ovarian steroid production during the luteal phase. The results suggest that plasma 17 beta-estradiol and progesterone determinations may serve as useful predictors for oocyte fertilizability in an in vitro system, following clomiphene-hCG stimulation and multiple oocyte recovery.

The probability of successful IVF outcome after poor ovarian response

Background. To evaluate the relationship between age and poor ovarian response to pregnancy and cancellation rate after IVF stimulation. Methods. Poor ovarian response was defined as ≤5 follicles at ovum pick up (OPU). Out of 1,706 consecutive OPUs performed during 2003/2004, 290 poor responders were identified. This cohort of poor responders was divided into two groups, ‘older’ and ‘younger’, with a cut‐off at the median age of 37 years. The pregnancy and cancellation rates after OPU were analysed. Results. Women aged >37 years, who were poor responders, had a significantly lower pregnancy rate per OPU (3.0%) compared to normal to high responders in the same age group (22.1%, p<0.05). Also, 43.6% of women >37 years, who were classified as poor responders, did not receive an embryo transfer (ET), compared to 13.2% of normal to high responders in this age group (p<0.05). Poor responders who were ≤37 years had a significantly lower pregnancy rate/OPU compared to normal to high responders who were ≤37 years (14.0 versus 34.5%, p<0.05) and a higher cancellation rate (40.1 versus 10.5%). Logistic regression analyses showed strong correlations between treatment outcome and the number of oocytes, age, and the mean and cumulative FSH dose given. The variable with the strongest impact on negative outcome was an interactive term between mean age and mean‐FSH dose (p = 5.0×10−5), indicating that women >37 years, who receive high doses of FSH have a significantly poorer treatment outcome than can be explained by either age or FSH dose alone. Conclusions. Poor ovarian response after IVF stimulation requires thorough counselling prior to OPU, regardless of the womans age. In poor responders >37 years of age, especially those who require high FSH doses, we suggest that the decision whether or not to proceed to OPU should include the couple after thorough counselling, even though the chance of successful outcome is extremely low.

Filled bladder simplifies human embryo transfer

Collection of pre-ovulatory oocytes for fertilization in vitro is now considered a quite straightforward procedure, especially in stimulated women and when using a teflon-coated aspiration needle. Moreover, cleavage of oocytes after fertilization in vitro has reached a high figure (Leeton et al. 1982). However, difficulties encountered in embryo transfer are not uncommon (Edwards et al. 1980; Leeton et al. 1982). Various technical procedures have been tried to facilitate the passage of the transfer catheter through the cervical canal into the uterine cavity. These techniques include placing the patient in different positions (Leeton et al. 1982; Jones et al. 1982), an outer cannula in the cervical canal (Leeton et al. 1982), dilatation of the cervical canal (Lopata et al. 1980), and the use of a tenaculum on the anterior lip of the cervix (Leeton et a]. 1982). Moreover, sedation of the patient has been used (Leeton et al. 1982), as some of these techniques can be uncomfortable for the patient and the procedure is occasionally protracted, while some can be unnecessarily traumatic. A simple technique is described for easy passage of the catheter into the uterine cavity in women who have an anteverted uterus. It is the angle between the cervical-uterine canal and the vagina (speculum exposed) that often makes it difficult for the transfer catheter to pass smoothly into the uterine cavity. A filled bladder will tilt the uterus to an upright position in line with the vagina (speculum exposed) (Figs 1 & 2). The woman is therefore asked to drink 1 litre of water 1 h before embryo transfer (as for an ultrasound examination). If ultrasound is used, the catheter can be seen entering the uterine cavity. Otherwise, ultrasound examination before embryo transfer is useful, to measure the exact length from the external 0 s to the fundus for controlled deposition of a transferred embryo (Leeton et al. 1982). In 14 consecutive embryo transfers, the filledbladder method was used. The woman was placed in a slight Trendelenburg position and the cervix was exposed with a bivalve speculum. An intrauterine device inserter was gently introduced 0.5-1.0 cm into the cervical canal to guide the

Ultrastructural characteristics of human oocytes fixed at follicular puncture or after culture

The material consisted of 73 oocytes obtained at follicle aspiration in ovarian-stimulated women. Oocytes in various stages of maturation were either immediately fixed or cultured before fixation. Observations by transmission electron microscopy disclosed that 20% of the immature oocytes, which appeared normal in the light microscope, had commenced atresia, and one immediately fixed oocyte surrounded by a normal-appearing preovulatory cumulus mass was found to be degenerated. Further, a cumulus mass judged as preovulatory contained an oocyte possessing a germinal vesicle. Light microscopy is thus not always adequate for judging the condition of ova surrounded by cumulus cells. Cytoplasmic changes which were regarded as being related to oocyte maturation were a decrease in the number of vacuoles and multivesicular bodies, an increase in the volume of the endoplasmic reticulum, a clustering of mitochondria, and the appearance of aggregates of tubuli of the smooth endoplasmic reticulum surrounded by mitochondria and mitochondria-vesicle complexes. Certain features were found to cause misinterpretations at examination of oocytes in a light microscope. For instance, polar bodies can be mimicked by corona cells in the perivitelline space and pronuclei in oocytes by large mitochondria-vesicle complexes in the ooplasm. Consequently, not all oocytes in which polar body-like or pronuclei-like structures are observed are necessarily fertilized oocytes.

Human oocytes express murine retroviral equivalents

Unfertilized human oocytes expressed a gp70-related epitope as observed when staining section immunocytochemically with a panel of monoclonal antibodies against gp70 of murine leukemia virus. Some oocytes also expressed virus-like particles at the cell membrane. Follicular fluids, corresponding to these oocytes, contained p30- and gp70-related antigens, reverse transcriptase, and an increased titer of interferon. The three- to four-cell human cleavage stages did not contain the gp70-related epitope. It is concluded that human oocytes, but not early cleavage stages, express products that suggest the presence of an active endogenous retrovirus genome.

Cumulative delivery rate in an in vitro fertilization program with a single embryo transfer policy

Objective. To investigate the cumulative delivery rate for women completing three fresh embryo transfer cycles or giving birth after the first or second transfer cycle in an in vitro fertilization program with a single embryo transfer (SET) policy. Setting. Privately run infertility clinic in southern Sweden (Skåne region). Population. Four hundred fifty‐three consecutive, unselected public patients beginning an ovum pick‐up cycle between July 2002 and June 2004. Method. Retrospective, observational study. Main outcome measure: Cumulative delivery rates. Results. Of 370 women who completed the study with up to three fresh ET cycles (90% SETs), 244 women (66%) progressed to delivery. Among the deliveries were three sets of twins (1%), one dizygotic. Cumulative delivery rates showed significant differences relative to age <36 (71%) and ≥36 years (47%), and delivery rates relative to rank of ET cycle (first and second vs. third ET cycle – 35, 34, and 21% delivery rates, respectively). Conclusions. The 66% cumulative delivery rate is in accordance with rates in similar observational studies and in estimation studies, in which predominantly two embryos were used for transfer. The multiple delivery rate was low. We suggest that repeated SET cycles can be implemented on a large scale even in a primarily unselected patient population without compromising the outcome.

The Ability of Ultrasound to Determine the time for Harvesting Preovulatory Oocytes

Abstract. To achieve fertilization in vitro, it would seem important to harvest the oocytes just before ovulation. Monitoring of follicular growth by repeated ultrasonic examinations has been suggested as a method for determining the time for harvesting preovulatory oocytes. To assess the usefulness of this new method, 90 women were studied in three groups. Two groups (N = 71) received ovarian stimulation with clomiphene citrate/hCG or hCG alone; one group (N = 19) was untreated. Each woman was examined with ultrasound 2–3 hours before the operation, performed on day 14 of the menstrual cycle. Blood samples for progesterone analyses were obtained from day 11 to 17 of the menstrual cycle to determine ovulation day. In the unstimulated group, the operation did not coincide with the day of the progesterone rise in any of the women and no preovulatory oocytes were found. In the stimulated women, progesterone rise and operation day coincided in 42%; preovulatory oocytes were obtained in 45% of the women. Follicles aspirated on the day of the progesterone rise were, on average, largest in diameter (20 mm, range 14–28 mm) and volume (3.3 ml, range 1 — 8 ml). When the follicles were ≥20 mm in diameter and the rise in progesterone occurred on the day of the operation, preovulatory oocytes were found in 63% of the women. If ultrasound failed to visualize follicles in an ovary, the chance of obtaining a preovulatory oocyte was 5.5%. False ultrasonic results were obtained in 29% of the women. In stimulated cycles, no improvement in the success rate of obtaining preovulatory oocytes would have been obtained if the operation day had been selected on the basis of one ultrasonic examination alone, compared with routine operation at midcycle. This lack of improvement was due to the wide range of the diameters of preovulatory follicles and to false results of the ultrasonic examinations. The investigation showed that the optimum time for harvesting fertilizable oocytes cannot be determined by ultrasound alone.

Some clinical results of in vitro fertilization by the Malmö group, Sweden

In our in vitro fertilization program, only clomiphene/human chorionic gonadotropin-stimulated cycles have been used, yielding 1.6 preovulatory oocytes/woman at follicle aspiration. The cleavage rate was significantly influenced by the percentage of abnormal spermatozoa and by the percentage of motile spermatozoa in the spermiograms. At embryo replacement, a tilted uterus obtained by a full bladder in the woman simplifies the replacement procedure. In the last 12 months, 47 embryo transfers have been carried out, resulting in five clinical pregnancies. Of these pregnancies, one has proceeded to term, one resulted in a miscarriage, and three resulted in ectopic pregnancies.

Scanning electron microscopy of human preimplantation endometrium in normal and clomiphene/human chorionic gonadotropin-stimulated cycles**Supported by the Swedish Medical Research Council (Project 12X-70) and the “Expressen” Prenatal Research Foundation.

Embryo transfer after in vitro fertilization of oocytes obtained from unstimulated or stimulated women is usually carried out earlier than when an embryo developed in vivo reaches the womb. The possible asynchrony between the developmental stage of the embryo and that of the endometrium at transfer might reduce the prospects for implantation; so also might any secondary effect of stimulation. Preimplantation endometrium dated by plasma hormone analyses of 17 beta-estradiol and progesterone in five unstimulated and eight stimulated women was surveyed. Three features were examined, namely, the frequency, distribution, and appearance of ciliated cells, nonciliated cells, and apical protrusion. Wide regional variations were observed, but no continuous or consistent changes or differences were found from ovulation to the time for implantation in unstimulated and stimulated cycles. Therefore, from the morphologic point of view, the surface might be ready to accept a transferred embryo at any time within a range of days after ovulation.