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.

The predictive value of the zona-free hamster egg penetration test in relation to in-vitro fertilization at various insemination concentrations.

The aim of the study was to evaluate the predictive value of the zona-free hamster egg penetration test (ZHEPT) for success in in-vitro fertilization (IVF) at various insemination concentrations ranging between 0.1 and >0.6 x 10(6)/ml. The ZHEPT was assessed using sperm samples from 87 couples undergoing IVF treatment. A similar test was simultaneously performed on the same semen sample following ionophore induction of the acrosome reaction (ZHEPTii test). Both the tests were poorly correlated with the fertilization rate of IVF at all the insemination concentrations except at >0.6 x 10(6)/ml, when there was good correlation between the ZHEPTii test and the fertilization rate. Following exclusion of two cases with an oocyte problem, further statistical analysis revealed that both the ZHEPT and ZHEPTii tests were poorly correlated with fertilization rate in IVF in this treatment group. This study suggests that the ZHEPT (with and without ionophore induction of the acrosome reaction) has a poor predictive value for the success of fertilization in IVF treatment at any insemination concentration.

Increased polymorphonuclear granulocytes in seminal plasma in relation to sperm morphology.

Much controversy surrounds the clinical significance of an increased concentration of white blood cells (WBC) in the male ejaculate. The World Health Organization's classification of leukocytospermia is a concentration > 1 x 10(6) WBC/ml. The aim of this study was to assess the association of varying concentrations of leukocytes to sperm morphology evaluated by strict criteria. Semen samples were collected from a total of 79 patients. Round cells on the initial semen analysis were stained for identification of polymorphonuclear granulocytes (PMN) as the largest group (50-60%) of white blood cells using the Endtz Method commercially produced as Leucoscreen. Diff Quick Staining Kit was used for sperm morphology assessment and 200 spermatozoa were assessed per slide. Data were evaluated using two cut-off criteria, at 0.5 x 10(6) WBC/ml and 1 x 10(6) WBC/ml. Mann-Whitney U-values at both < and > 0.5 x 10(6)/ml PMN (P < 0.001) and at < and > 1.0 x 10(6)/ml PMN (P < 0.015) showed differences between percentage normal forms. Spearman's rank correlation coefficient for PMN concentration showed a negative correlation (P < 0.01) with percentage normal sperm morphology and positive correlation for midpiece abnormalities (P < 0.04). These data support the hypothesis that PMN have a role in the increase of abnormal spermatozoa, particularly those with midpiece abnormalities, by as yet unknown mechanisms.

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.

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)

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%.

Implantation, abortion, and birth after in vitro fertilization using the natural menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin.

The incidence of pregnancy and abortion was analyzed in 1679 patients having embryos replaced after oocyte recovery and in vitro fertilization in order to alleviate their infertility. In these patients, 364 pregnancies were achieved and 108 abortions occurred. Patients were treated either on their natural cycle, having a spontaneous luteinizing hormone (LH) surge to induce ovulation, or after ovarian stimulation using clomiphene citrate alone or in combination with human menopausal gonadotropin (hMG). The data were assessed in relation to the numbers of embryos replaced, the follicular stimulation and ovulation induction regime used, the dose of gonadotropins and the dose of antiestrogens, and the age of the patient. The most successful treatment for the number of live births per laparoscopy was the use of clomiphene citrate in combination with human menopausal gonadotropin followed by human chorionic gonadotropin (hCG) to induce follicular maturation and ovulation. This treatment produced a significantly higher (P less than 0.001) number of patients with multiple embryos (86.5%). There was no significant effect on pregnancy or abortion with a low (less than 400-mg) or high (greater than or equal to 400-mg) dose of clomiphene. The total dose of gonadotropins used did not influence the incidence of pregnancy or abortion. The use of hCG with hMG induced a significant (P less than 0.01) positive effect on the incidence of pregnancy. The incidence of pregnancy showed a progressive decline with increasing age but there was a highly significant (P less than 0.01) increase in the incidence of abortion with increasing age. With increasing numbers of embryos replaced, up to three, the incidence of deliveries increased.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

Treatment of male infertility by in vitro fertilization: factors affecting fertilization and pregnancy.

IVF was attempted in more than 100 couples where the man was considered to be the cause of the infertility. 57% of all oocytes were fertilized and embryo replacement was achieved in 60-70% of cases. Pregnancies following IVF were established in cases with a long duration of infertility and different male factors such as oligospermia, teratospermia, asthenospermia and auto-immunity. Almost 50% of men with very low numbers of active spermatozoa (0.5 X 10(6)/ml motile spermatozoa) were able to establish pregnancy. No correlation was found between the percentage motility and the chance of fertilization in cases with abnormal semen, but a reduction of the incidence of fertilization was noticed in cases with extreme oligospermia (5 X 10(6)/ml) where 41% of the oocytes were fertilized. The results of the post coital hamster egg tests were inaccurate in predicting the outcome of IVF. The collection of split ejaculates, and the careful preparation of spermatozoa using sedimentation and layering methods proved to be beneficial, improving sperm motility and raising the chance of fertilization.

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.

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.

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.