Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro.

Human oocytes were collected by laparoscopy and fertilized and cultured in vitro. Human chorionic gonadotropin was detected in the medium surrounding two embryos cultured for more than 7 days after fertilization.

The influence of transient hyperprolactinemia on in vitro fertilization in humans.

Hormonal stimulants of ovarian follicular maturation and anesthesia/surgery were examined for their effects on the concentration of plasma and follicular fluid PRL. Forty-seven patients undergoing in vitro fertilization for the treatment of infertility were selected at random for this prospective study. Patients given human menopausal gonadotropin and clomiphene citrate had significantly higher levels of plasma PRL compared to those given clomiphene only. Anesthesia/surgery elevated plasma PRL levels in all patients, by as much as 50-fold and to as high as 7878 mIU/liter. Follicular fluid PRL levels were correlated with preanesthetic plasma PRL concentrations, but the latter were not correlated with plasma 17 beta-estradiol. Elevated plasma or follicular fluid PRL concentrations had no effect on in vitro fertilization of oocytes or embryonic development. Although not significant, the incidence of pregnancy was highest in the group of patients with the lowest preanesthetic plasma PRL levels.

Follicular steroids as a prognosticator of successful fertilization of human oocytes in vitro.

Oocytes were collected by aspiration of preovulatory follicles from 55 women. The preovulatory rise in LH was monitored in urine using the Hi-Gonavis (Mochida Pharmaceuticals) technique. Patients were treated either during the natural cycle or after the induction of ovulation with clomiphene citrate. After collection and culture, the oocytes were inseminated with the spermatozoa of the husband. The levels of progesterone, oestradiol-17 beta and androstenedione in the clear follicular fluid were measured by radioimmunoassay. A multivariate analysis containing these three hormone levels together with two ratios of progesterone with each of the other hormones indicated reasonable discrimination between the oocytes which fertilized and those which remained unfertilized after insemination. The discriminant analysis suggested that the fertilization of the oocytes could have been predicted on the basis of these hormonal profiles with a success rate which exceeded 90%.

Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.

In all, 1210 treatment cycles were divided into three categories for retrospective analysis according to the period of delay between oocyte retrieval (occurring at a fixed time after human chorionic gonadotrophin) and intracytoplasmic sperm injection (ICSI) of <3 h, 3-5 h, >5 h (referred to as 'delayed ICSI'). Three stages from oocyte to the birth of a live baby were identified for statistical analysis, (i) fertilization (2PN zygotes), (ii) cleavage of 2PN zygotes, (iii) transferred embryo to live birth. Stages 1, 2 and 3 were analysed statistically for the three time periods. Chi-square analysis showed no significant effect of delayed ICSI on fertilization (chi(2) = 3.615, P = 0.65), and embryo transfer to birth (chi(2) = 1.840, P = 0.399). The effect on cleavage was significant (chi(2) = 9.625, P = 0.008). However, shorter incubation times produced results which were better than the traditional longer ones. The success rate at the cleavage stage was so high that the marginal advantage had very little effect on the overall process. This study of a substantial patient sample establishes that ICSI on a peri-ovulatory oocyte (<3 h after oocyte retrieval) does not compromise outcome parameters, and that longer periods of incubation (>5 h) do not offer a statistically significant advantage.

Analysis of 25 infertile patients treated consecutively by in vitro fertilization at Bourn Hall.

Twenty-five infertile women suffering from tubal disorders were treated consecutively by in vitro fertilization over a 14-day period. Follicular growth in 24 of them was stimulated with clomiphene citrate, ovulation being induced by an endogenous surge of luteinizing hormone or an injection of human chorionic gonadotropin. One patient was given tamoxifen and had an endogenous luteinizing hormone surge. One or more oocytes were fertilized, and at least one embryo was replaced in 19 patients. Nine pregnancies were established, and eight infants have been delivered, a pregnancy rate of 36% per laparoscopy and 47% per replacement. A detailed analysis of each patient is presented.

Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study.

The survival and implantation capacity of cryopreserved cleaving (5-cell to 10-cell) human embryos and expanded blastocysts was compared. Twice as many cleaving embryos were frozen as were expanding blastocysts because of the low developmental potential of human embryos in vitro. However, significantly more expanded blastocysts survived cryopreservation than cleaving embryos, and relatively more pregnancies were established by the replacement of thawed blastocysts than by the replacement of thawed cleaving embryos. Cleaving embryos from 26 women were thawed; 17 had thawed embryos replaced, and 4 subsequently became pregnant. Expanded blastocysts were thawed from 23 other women; 15 had thawed blastocysts replaced, and 8 subsequently became pregnant. The pregnancy of one patient in each group aborted; both patients were over 40 years of age. It is estimated that by maintaining the current policy of replacing three fresh embryos and freezing any remaining embryos when they reach blastocyst stage, the total incidence of pregnancy would increase by 3%.

In vitro fertilization using cryopreserved donor semen in cases where both partners are infertile.

The incidence of pregnancy after in vitro fertilization (IVF) was studied in a group of 38 couples (55 cycles) where both partners were infertile. Cryopreserved donor semen (IVF-D) was used in all cycles. Results were compared with those in a control group of couples where the husband's semen was considered normal and only the wife was infertile. No significant differences were found between the IVF-D and control groups in the incidence of fertilization (80% versus 72%), pregnancy per cycle (33% versus 29%), and abortion (18% versus 20%), despite the considerably lower percentage of motile spermatozoa in the IVF-D group. Forty percent of patients, each treated unsuccessfully with at least 12 artificial inseminations with donor semen, became pregnant after one or two IVF-D cycles. It is concluded that IVF with frozen donor semen is a beneficial treatment for couples where both partners are infertile.

Oocyte maturation and fertilization in vitro.

From the time of early follicular growth to some time after ovulation, the oocyte undergoes many critical changes in its biochemistry. In this paper the author describes different steps in oocytes maturation some of which, during the early period, will require support from the follicle cells, while later in development endogenous synthetic activity can be maintained in the absence of such support.

Analysis of mouse uterine proteins at pro-oestrus, during early pregnancy and after administration of exogenous steroids.

Uterine secretions were analysed after labelling with L-14,5-3H]leucine. During pro-oestrus major protein peaks were seen with approximate mol. wt 2.0 X 10(4), 2.7 X 10(4), 4.0 X 10(4), 5.7 X 10(4), 7.8 X 10(4) and 13.0 X 10(4). Qualitative changes were observed during the first 4 days of pregnancy. On day 4 p.c. secretions revealed two prominent peaks with mol. wt 6.7 X 10(4) and 12.5 X 10(4), four peaks between 2.0 and 4.7 X 10(4) and one at 8.5 X 10(4) and one at 20.0 X 10(4). Profiles similar to those on mimic Day 4 p.c., and implantation, were induced in ovariectomized females by administration of exogenous progesterone and oestradiol, whereas the proteins detected after administration of exogenous oestradiol alone were essentially similar to those found at pro-oestrus. Ovariectomized mice treated with the steroid sequence to mimic Day 4 p.c. profiles but not given the final injection of oestradiol showed no detectable labelled proteins. Injection of progesterone or oestradiol alone, in the final sequence of injections given to ovariectomized pregnant females treated with progesterone, did not induce blastocyst implantation. These studies indicate a synergistic relationship between progesterone and oestrogen in the secretion of uterine macromolecules.

Radiolabelled uterine proteins during early pregnancy and pseudopregnancy in mice after unilateral ovariectomy and superovulation.

Uterine secretions were analysed after labelling in vivo with L-[4,5-3H]leucine. The acid-precipitable counts were highest on Day 1 p.c., low on Day 2, lowest on Day 3, and similar to Day 2 values on Day 4 p.c. After unilateral ovariectomy the acid-precipitable counts for the horn ((+)ovary) ipsilateral to the remaining ovary were higher than those in the contralateral horn ((-)ovary) for each condition studied. Superovulation yielded slightly higher acid-precipitable counts in the (-)ovary and (+)ovary horns of pregnant mice on Day 3 p.c., and in the (+)ovary horn only for pseudopregnant mice on Day 2 p.c. The Day 2 counts for the (+)ovary and (-)ovary horns of the superovulating pseudopregnant mice were higher counts than those for superovulating pregnant mice, but the reverse occurred for (+)ovary horns only on Day 4 p.c. No qualitative differences between pseudopregnant and pregnant mice were observed on Day 4 and superovulation appeared to enhance the profiles obtained.

The role of divalent cations in the metabolic response of mouse blastocysts to serum.

Uptake and incorporation of [3H]uridine by mouse blastocysts was measured in response to serum under various conditions of Ca2+ and Mg2+ availability. Previous studies (Fishel & Surani, 1978) showed that serum stimulated uptake and incorporation of uridine in blastocysts. Here it is shown that in Ca2+-deficient medium (< 20 microM-Ca2+) cell metabolism was also inhibited and increasing [Ca2+] resulted in increased uptake and incorporation. Maximum stimulation required an extracellular [Ca2+) of 0.25 mM. The effects of low Ca2+ were reversible and could also be alleviated by 15 and 20 mM-Mg2+. Magnesium greater than 20 mM was deleterious. Inorganic phosphate (Pi) was used to complex free Mg2+ in order to maximize the effects of Mg2+ deficiency. Inhibition was reversed by increasing [Mg2+] or by 15-20 mM-Ca2+. Calcium concentrations greater than 20 mM inhibited maximum stimulation. Inhibitors of Ca2+ influx D600 and papaverine, inhibited stimulation at concentrations above 5 and 20 microM respectively. Magnesium concentration of 15 mM alleviated the inhibitory effects of 50 microM D600. The effect of Ca2+-deficient medium was also alleviated by Sr2+. The results suggest that both Ca2+ and Mg2+ are required for blastocysts to respond maximally to serum; their initial role appeared to involve the binding of stimulatory serum molecules to the cells of the blastocyst followed by an influx of these cations.

Morphology and X-ray microprobe analysis of spermatozoa from fertile and infertile men in in vitro fertilization.

This paper reports differences observed in the elemental content of fertile and infertile human spermatozoa used in an in vitro fertilization (IVF) program. "Fertile" and "infertile" were designated by the successful penetration or failure to penetrate an oocyte in vitro. We report morphological and morphometric differences which, together with elemental changes, may be causes of infertility in apparently normal spermatozoa. There were significant differences (P less than 0.05) in sodium and chlorine concentrations between fertile and infertile samples and there was more chlorine than could be accounted for as sodium chloride. Many spermatozoa showed particles adhering to tails, with a higher incidence of "contamination" in the infertile spermatozoa. There were significant differences in both shapes of heads and lengths of tails between fertile and infertile spermatozoa.

Embryonic and uterine factors in delayed implantation in rodents.

An hypothesis for delayed implantation and embryonic quiescence is proposed on the basis of adaptation of blastocysts to changes in their environment. The environmental factors considered consist of macromolecules and essential ions and metabolites such as Ca2+, Mg2+ and glucose. Macromolecules appear to induce an influx of these essential metabolites which is followed by metabolic enhancement in blastocysts in the absence of any of the essential factors, the initial stages of embryonic diapause follow. The prevention of influx of metabolites such as Ca2+ by specific inhibitors, D600 or papaverine, also prevents activation of blastocysts. Early cleavage stage embryos show little or no response to changes in environmental macromolecules which may explain why embryos usually enter into quiescence at the blastocyst stage when the environmental constraints on blastocyst development become very marked. This also coincides with termination of cleavage and initiation of cell growth in embryos. The increase in responsiveness of embryos is attributed to several inherent changes including cell surface and functional changes in the plasma membrane. The conditions for embryonic quiescence in vivo are not species specific. Trophoblastic vesicles without the inner cell mass can also enter into quiescence. Studies using the antibiotic, tunicamycin, which inhibits protein glycosylation and prevents trophoblast adhesion and giant cell outgrowths, suggest that the cell surface interactions may involve glycoproteins. Such interactions may be crucial for implantation as well as for maintaining embryos in diapause for prolonged periods of time. A short sojourn in diapause for certain blastocysts which do not normally develop to an advanced stage, appears to have a beneficial effect on subsequent development. The overall significance of this suggestion for other species showing obligatory diapause is unclear.

Evidence for the synthesis and release of a glycoprotein by mouse blastocysts.

Mouse blastocysts were incubated in microdrop cultures in the presence of D-[6-3H]glucosamine. The embryos and the incubation medium were analysed for the incorporation of label into acid-precipitable material. Of the precursor incorporated into blastocysts, 8-13% was detected in the medium. Several glycoproteins were found in the blastocysts and one of molecular weight about 87 000 was also detected in the extracellular medium. In the presence of tunicamycin, the amount of the labelled glycoprotein was reduced by 30-40% in the embryos and 47-69% in the medium. However, the same relative proportion of label was still detected in the extracellular fraction of 87 000 mol. wt. The study demonstrates the synthesis and release of a glycoprotein by mouse blastocysts, the mechanism of which is tunicamycin-insensitive.

Changes in responsiveness of preimplantation mouse embryos to serum.

Changes in uptake of radioactive uridine and its incorporation into RNA were determined in preimplantation mouse embryos, from the 2-cell to the blastocyst stage, as a measure of their responsiveness to extracellular conditions. Two media were tested, one contained serum and the other contained bovine serum albumen as a control. An increase in the acid-soluble pool occurred at the 8-cell stage and a marked increase in RNA synthesis occurred at the early blastocyst stage when the embryos were incubated with serum.

The application of the zona-free hamster egg test for the prognosis of human in vitro fertilization.

The zona-free hamster egg test was carried out using spermatozoa from 15 men which consistently failed to fertilize their wives' oocytes in vitro. Spermatozoa from nine of these men fertilized hamster eggs in vitro, indicating that positive results in this assay are an unreliable guide to human in vitro fertilization. Donor spermatozoa were needed to fertilize the wife's oocytes in three of these cases. Nevertheless, the proportion of hamster egg penetration was significantly lower compared with spermatozoa from 15 men who could fertilize their wives' oocytes in vitro. The hamster assay also failed to indicate the establishment of pregnancy.

Pregnancies following the frozen storage of expanding human blastocysts.

Human blastocysts were frozen in Earle's solution containing pyruvate and human serum, using glycerol as cryoprotectant, and stored in liquid nitrogen. Thawed blastocysts were replaced in 11 patients, which resulted in two pregnancies. One blastocyst giving a pregnancy was hatching when replaced. Three parameters appeared to be important for embryo survival and implantation: the interval between ovulation and replacement of the thawed blastocysts, satisfactory embryonic development before freezing, and the stage of blastulation when cooling began.

Establishing pregnancies after follicular stimulation for IVF with clomiphene citrate and human menopausal gonadotrophin only.

Data are presented on establishing pregnancies by IVF during 1987 using only clomiphene citrate and human menopausal gonadotrophin for follicular stimulation. Of the 562 patients undergoing follicular stimulation, 80% reached oocyte recovery and 70% had at least one conceptus replaced. Patients having one or more (up to a maximum of four) conceptuses replaced demonstrated a significant increase in the establishment of pregnancies from one to two (14-29%: P = 0.035) and from two to three conceptuses (29-42%: P = 0.037). There was a significant decline in pregnancies when four conceptuses were replaced compared with three (P = 0.004). The data were also analysed according to the cause of infertility, specifically tubal, endometriosis, unexplained infertility and male factors only. After the replacement of conceptuses, the incidence of implantation and abortion was not significantly different. The incidence of pregnancy declined significantly after 35 years (26%) compared with women under 31 years (43%; P = 0.043). Of 129 women having three conceptuses replaced, in those greater than 35 years (63 patients) 23 (37%) became pregnant whereas in those less than 31 years (65 patients), 34 (52%; P = 0.05) became pregnant. Twenty-two per cent of stimulated cycles resulted in an endogenous LH surge and the incidence of patients having three conceptuses replaced in this group was lower than those in the HCG group (P = 0.007). Fertilization per oocyte was also significantly reduced (P less than 0.001) in patients with an LH surge. In total, 2824 oocytes were recovered and 57% fertilized with 54% of patients having three conceptuses replaced.(ABSTRACT TRUNCATED AT 250 WORDS)

Human sperm morphology evaluation pre- and post-Percoll gradient centrifugation.

Previous experiments have established a relationship between the morphological characteristics of human spermatozoa and their fertilizing potential in vitro. To assess further the efficiency of Percoll gradient centrifugation (PGC) as a method of sperm selection, we have examined morphological characteristics of spermatozoa from 86 teratozoospermic patients attending Nottingham University Research and Treatment Unit in Reproduction (NURTURE). Patients were divided into groups according to percentage normal morphology in the fresh sample: group A (n = 14), < 5% normal morphology; group B (n = 41), 5-14% normal morphology; and group C (n = 31), > 14% normal morphology. Morphology slides were prepared using Diff Quik staining techniques and evaluated by Kruger strict criteria, under oil immersion, at a magnification of x1000; specific defects, viz. head, neck, cytoplasmic droplets, tail, immature cells, were assessed individually. Following PGC, a sperm sample with enhanced morphology was recovered for group B (P < 0.01) and C (P < 0.005); however, for group A (very severe teratozoospermia) PGC did not select a sample with significantly improved morphological quality. Specific sperm defects affected by PGC were head, neck and immature cells. No significant difference was found for tail abnormalities or cytoplasmic fragments.