Graham M. Warnes

Culture factors in relation to the success of human in vitro fertilization and embryo transfer

The development of 1-cell mouse zygotes to the blastocyst stage in vitro has been used as a quality control for the media employed for human in vitro fertilization and embryo transfer (IVF-ET). The following procedures were associated with high rates of mouse embryo development and human pregnancies following IVF-ET: adequate gassing and equilibration of the medium, double rinsing of pipettes and catheters used to handle embryos, use of a HEPES-buffered medium for manipulating embryos in the absence of an atmosphere containing 5% CO2, control of excessive temperature in the vicinity of the embryos, and ET using medium containing 50% patients serum. The institution of these procedures gave more consistent pregnancy rates. However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by the mouse embryo development test.

MORPHOLOGICAL AND FUNCTIONAL RELATIONS OF GRAAFIAN FOLLICLE GROWTH TO OVULATION IN WOMEN USING ULTRASONIC, LAPAROSCOPIC AND BIOCHEMICAL MEASUREMENTS

The daily growth rates of ovarian follicles were recorded ultrasonically for five days until ovulation in 56 spontaneously ovulating women and related to endocrine and clinical parameters. Over the 5‐day period, the average diameter of the follicle destined to ovulate increased from 12 to 23 mm, the second largest follicle from 6 to 12 mm, the third largest follicle from 5 to 9 mm and the fourth largest follicle from 4 to 8 mm. Similar but lesser growth rates occurred in the follicles in the contralateral ovary. Ovulation occurred within 24 hours of the luteinizing hormone (LH) peak, and the mean peak diameter of the ovulating follicle was 23.2±0.3 (SEM) mm, (range 18–29 mm) before ovulation, and subsequent luteal function was judged to be normal. Follicular growth was most closely correlated with increasing peripheral blood oestrogen levels. In 16 women who had a laparoscopy within 12 hours of the last ultrasound and following the LH peak, the mean diameter of the largest follicle as measured by ultrasound (23.6±0.4 mm) was similar to that measured at laparoscopy (22.8±0.4 mm) and estimated from the volume of follicular fluid aspirated (average 5.8±0.2 ml), 22.5 mm. The follicular fluid levels of progesterone were high on the day of the LH peak and blood progesterone levels had risen significantly indicating that luteinization of the dominant Graafian follicles had already occurred prior to ovulation. This study confirms that ultrasonic monitoring provides a reliable measure of follicular growth and allows studies correlating morphological changes with both normal and abnormal endocrine function of the human ovary.

Failure of body mass index or body weight to influence markedly the response to ovarian hyperstimulation in normal cycling women

A retrospective analysis was performed of 368 normally cycling women treated with a single cycle of a standard ovarian hyperstimulation regime (CC 100 mg days 5 to 9 and hMG 150 IU days 6, 8, and 10) associated with either an IVF or GIFT program. Neither the peak serum E2 level attained nor the number of days of stimulation required bore a relationship to the BMI or the total body weight of these women. Whereas the mean number of oocytes aspirated from women with BMI less than 19.1 was higher (6.4 +/- 3.2) compared with obese women (BMI greater than 27.6, 4.8 +/- 2.6), the rate of fertilization was not different for both BMI extremes. It is concluded that factors other than BMI or total body weight have more important influences on the response to hyperstimulation in normal women.

Pregnancy-related chemotactic activity of human follicular fluid *

We measured chemotactic activity in 238 follicular fluids (FF) aspirated from 45 women who had undergone ovarian stimulation with a combination of clomiphene citrate and human menopausal gonadotropin for oocyte retrieval, in vitro fertilization, and embryo transfer. Fifteen of the treatment cycles resulted in pregnancy. The mean chemotactic activity, measured as the distance in microns granulocytic leukocytes migrated through a 3.0-micron membrane, was significantly higher in FF from conceptual cycles, compared with nonconceptual cycles. Serum chemotactic activity was significantly lower in conceptual cycles, compared with nonconceptual cycles. A chemotactic gradient appears to exist between the peripheral circulation and the ovarian follicle. The gradient favors the follicle in conceptual cycles, as indicated by the chemotactic quotient (the ratio of chemotactic activity of FF to serum). In conceptual cycles the chemotactic quotient was 1.7 +/- 0.17, compared with 0.7 +/- 0.03 for nonconceptual cycles. The presence of leukocyte chemotactic factor in FF appears to discriminate prospectively with a 90% degree of confidence between conceptual and nonconceptual in vitro fertilization and embryo transfer cycles.

Human luteal phase function following oocyte aspiration from the immediately preovular graafian follicle of spontaneous ovular cycles.

Human luteal phase function as evaluated by peripheral venous blood steroid levels does not appear to be impaired following the aspiration of follicular fluid together with a cumulus enclosed oocyte and a number of granulosal cells from the immediate preovular follicle in women having otherwise spontaneous ovular cycles. The day to day levels of luteinising hormone, oestradiol‐17β, 17α‐hydroxyprogesterone, progesterone and basal temperatures in 14 women who had their preovular follicle aspirated were compared with a control group of 28 spontaneously ovulating women. It was concluded that a carefully performed single aspiration of the contents of a preovular follicle, for the purpose of extra‐corporeal fertilisation of the mature oocyte, did not lead to impaired steroid function of the subsequent corpus luteum, although the prolactin levels were increased due to the effects of the relaxant anaesthetic and/or the laparoscopic procedure. A safe and simple laparoscopic procedure is also described, which is particularly suitable for women with a likelihood of extensive pelvic adhesions.

In vitro fertilization and embryo transfer program, Department of Obstetrics and Gynecology, University of Adelaide at the Queen Elizabeth Hospital, Woodville, South Australia

A review is presented summarizing the in vitro fertilization experience at the University of Adelaide, Australia. Initial attempts utilizing the normal cycle were unsuccessful in obtaining a pregnancy. Since 1982 the overall ongoing pregnancy rate has been 21% per embryo transfer, 16% per laparoscopy, and 12% per treatment cycle. A detailed description of presently utilized methodology is presented.

Endocrinology of Ovarian Stimulation for In Vitro Fertilization

Summary: Understanding of the endocrinology of in vitro fertilization has advanced rapidly in the past 5 years. Despite a multitude of ovarian stimulation regimens a particular regimen has not demonstrated a marked superiority. In principle the achievement of high FSH levels during the early follicular phase to recruit a maximum number of follicles with a tolerable degree of asynchrony for final maturation is confined to a limited time span or ‘FSH window’ of about 3 to 4 days before negative E2 feedback induces below‐threshold FSH levels, thereby condemning all subsequent follicles in that cycle to atresia. It appears that one can widen and/or amplify the ‘FSH window’ too far from gross hyperstimulation resulting in the recruitment of many follicles with an intolerable degree of asynchrony. This may lead to a defective endocrine environment for the oocytes contained within these follicles or an abnormal luteal environment and an increased frequency of fertilization, cleavage and implantation failure.

Decrease in luteal gonadotropin concentrations in conception cycles after in vitro fertilization/gamete intrafallopian transfer

The concentrations of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured in the luteal phase of the cycle in patients undergoing ovarian hyperstimulation. In nonconception cycles, FSH and LH were increased in the late luteal phase compared with conception cycles in which both gonadotropins were suppressed. Estradiol (E2) and progesterone concentrations increased in pregnancy cycles and may be the sole cause for the decreased gonadotropin concentrations as shown by equivalent concentrations of LH and FSH in both pregnancy and nonpregnancy cycles after matching for E2 concentrations. Subjects who subsequently had twin pregnancy or a spontaneous abortion were compared with those with a successful ongoing singleton conception. There were no significant differences relative to LH and FSH between the three groups, although in twin pregnancy FSH tended to be lower at day 16 from oocyte recovery. It is concluded that suppression of LH and FSH in hyperstimulated pregnancy cycles occurs after the time of the rising human chorionic gonadotropin concentrations in plasma.

Successful Treatment of Severe Male Factor Infertility in 100 Consecutive Cycles Using Intracytoplasmic Sperm Injection

In this report, we present the results of our first 100 consecutive cycles of intracytoplasmic sperm injection (ICSI). Overall, fertilization occurred in 98% of cycles and embryos were transferred in 94% (2.6 embryos per cycle). About 50% of patients had embryos frozen. The overall fertilization rate was 71%, of which 4% were abnormally fertilized (three pronuclei). A total of 30 clinical pregnancies were established (32% per transfer), resulting in 18 singleton, six twin and one triplet ongoing pregnancies. The implantation rate per embryo was 15%. There were no significant differences in the fertilization or pregnancy rates between patients who had only occasional motile spermatozoa in the ejaculate, semen that was too poor for routine in-vitro fertilization (IVF), or who had failed routine IVF and/or subzonal sperm injection (SUZI). A group of 18 patients were treated with both ICSI and routine IVF on their first cycle because of the high likelihood of failed fertilization due to poor sperm morphology < 20% normal). In this group, ICSI oocytes had a fertilization rate of 76% compared to only 15% for the routine IVF (control) oocytes, and six patients conceived after transfer of ICSI embryos (33%), indicating that ICSI can be used successfully on 50% of the oocytes if fertilization failure is expected. Similarly, patients who had failed to become pregnant with SUZI achieved excellent results after ICSI. There were no significant differences between ICSI and routine IVF in the proportions of grade 1, 2 or 3 embryos on day 3 post-oocyte recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Ageing and fertility

To examine whether fecundity is affected by age, peripheral oestradiol (E2) measurement and reproductive potential were studied in patients in different age groups receiving ovulation induction prior to in vitro fertilization (IVF) and embryo transfer (ET). The term pregnancy rate of 179 patients who had reversal of sterilization following 4 different sterilization procedures was also assessed in similar age groups. In the IVF group, age did not influence maximal E2 rise prior to oocyte retrieval, number of oocytes available for fertilization, fertilization rate, miscarriage or clinical pregnancy rate up to the age of 40. Similarly, in the reversal of sterilization group, term pregnancy rate was not affected by age, but was affected by the method of sterilization originally performed.We conclude from this study that IVF and reversal of sterilization procedures may be successful in women in the later reproductive age group.