The purpose of this study was to investigate whether the endometrium of women with unexplained infertility differs in some immunological aspects from the endometrium of normal fertile women. Endometrial biopsies were obtained from 24 normal fertile women (group I) and 24 women suffering from unexplained infertility (group II) at 4, 7, 10 and 13 days following the luteinizing hormone (LH) surge. Endometrial granulated lymphocytes were assessed morphometrically in 2 microns resin sections. A panel of 11 monoclonal antibodies was employed to characterize the leukocyte subsets in frozen sections. Semi-quantification was performed with a Quantimet 970 image analyser. Data were analysed using one- and two-way analysis of variance. Compared with fertile controls, women with unexplained infertility had significantly lower numbers of CD8+ (T suppressor/cytotoxic) cells at each post-LH date. In contrast, the number of CD4+ (T helper/inducer) cells was significantly higher in group II. Throughout the luteal phase, infertile women had fewer CD56+ cells than normal fertile controls. The volume fraction of endometrium occupied by the nuclei of endometrial granulated lymphocytes did not alter with the cycle stage but the mean nuclear diameter and axial ratio decreased from LH+7 to LH+13. The differences observed in endometrial leukocytic subpopulations between fertile and infertile women may contribute to unexplained infertility probably by affecting the embryonic maternal dialogue during the implantation and early placentation period.

The physical interaction between human spermatozoa and the epithelium of the human uterine (Fallopian) tube was investigated in vitro using a variety of techniques. The 'live' observation of human spermatozoa incubated with 1 day old cultures of tubal epithelium demonstrated that spermatozoa can show a strong physical interaction with epithelial cells; contact with the epithelium appeared to be random and there was no evidence of any taxis toward epithelial cells. The physical interaction (or 'binding') was resistant to gentle washing and was maintained following the addition of glutaraldehyde fixative. The intimate nature of the interaction was confirmed ultrastructurally where both spermatozoa and epithelial membranes were observed to be in close apposition. These results are the first descriptions of sperm-epithelial 'binding' in the human. They are similar to other observations made in a variety of non-human mammalian species. It is suggested that this interaction may be an important feature of normal sperm transport in the human uterine tube in vivo.

No abstract

A simple co-culture bioassay system was used to investigate whether or not the anatomical origin affected the ability of epithelial cells from the human uterine (Fallopian) tube to 'bind' spermatozoa. This study was also used to identify some of the factors which may be involved in the regulation of sperm-epithelial interactions in vitro by comparing different tissue culture models and assessing the effect of oestradiol concentration. Epithelial explants harvested from different regions of human uterine tubes were co-incubated with a known concentration of motile donor spermatozoa. All results were adjusted to reflect a standard sperm concentration of 5 x 10(6)/ml. More spermatozoa associated per field of isthmic compared to ampullary epithelium [isthmus 9.5 +/- 0.9, ampulla 7.1 +/- 0.7 (mean +/- SEM); n = 36, P < 0.05, ANOVA] and cells from post-menopausal patients had an apparently reduced ability to bind spermatozoa [isthmus 5.5 +/- 2.0, ampulla 4.3 +/- 1.4 (mean +/- SEM); n = 4]. Neither menstrual cycle stage nor addition of mid-cycle concentrations of 17beta-oestradiol (750 pmol/l) affected the number of spermatozoa which bound to epithelium from either tubal region. In addition, the number of spermatozoa which bound per field of polarized explants was greater (P < 0.05) than that bound to dissociated primary and passaged epithelial cell monolayers. This report is the first to provide evidence suggestive of a role for sperm-epithelial binding in the formation of an isthmic sperm reservoir in the human uterine tube. Results also indicate that oestrogen is not involved in the regulation of these interactions, and that cell polarity is an important factor for such associations in vitro.

The behaviour of human spermatozoa was observed during incubation with epithelial cells isolated from the isthmic and ampullary sections of human uterine (Fallopian) tubes. During incubation, spermatozoa were observed to bind to the epithelial cells of the tube (the endosalpinx), and individual spermatozoa attached and detached at intervals. The kinematic characteristics of spermatozoa during these behaviour patterns were determined. The results showed that detached spermatozoa typically had an increased curvilinear velocity and amplitude of lateral head displacement, accompanied by a decrease in their linearity. Significantly (P < 0.01) more of the detaching spermatozoa were hyperactivated than were spermatozoa prior to attachment for both isthmic (35.3 +/- 5.5 versus 4.0 +/- 3.3%; mean +/- SEM) and ampullary (26.0 +/- 7.0 versus 2.0 +/- 1.4%) regions. Incubation with epithelial cells from either region produced no differences in any category of sperm behaviour. Furthermore, there was no significant difference between regions in the amount of time spermatozoa spent bound (33.6 +/- 12.9 and 20.6 +/- 3.0 s for isthmic and ampullary tissue respectively). These results support the hypothesis that hyperactivation may assist spermatozoa in breaking connections with epithelial cells.

Male rats undernourished from the 18th day of gestation until 100 days of age were nutritionally rehabilitated until 200 days of age. Six control and six experimental rats at each of 12, 25, 50, 100, and 200 days of age were killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscope levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. There were no statistically significant differences in the synapse-to-neuron ratio between control and undernourished rats at 12, 25, and 50 days of age. However 100-day-old undernourished rats had a significant deficit in this ratio compared to age-matched controls. Despite this, 200-day-old nutritionally rehabilitated rats were found to have, on average, 23% more synapses per neuron than controls. In both the control and the undernourished groups the synapse-to-neuron ratio increased to a peak by 50 days of age. This was followed by a significant fall in the ratio by 100 days of age. Although there was no further change in the control rats, the experimental group showed a substantial increase in the ratio by 200 days of age. This latter increase appeared to be related to the period of nutritional rehabilitation.

The concentrations of CA 125 and placental protein 14 (PP14) were measured in uterine flushings obtained throughout the luteal phase of the cycle from eight normal fertile women. The concentrations of both proteins increased in a similar pattern throughout the luteal phase of the cycle, with the most dramatic increase occurring 6 days after their luteinizing hormone surge (day LH +6). However, a greater variation in CA 125 concentrations was seen compared to that seen for PP14. The concentrations were compared to those obtained on day LH +7 of the cycle from a group (n = 35) of women with recurrent miscarriage. The ranges in concentration of PP14 and CA 125 in the flushings of fertile and recurrent miscarriage patients were very similar. However, a greater proportion of women with recurrent miscarriage (55%) had low concentrations (< 5 ng/ml) of PP14 than in the control group (12.5%) and the concentrations of PP14 in the uterine flushings were significantly less (P < 0.05) in women with recurrent miscarriage compared to the normal fertile group. There was no significant difference in the concentration of CA 125 in the uterine flushings between the two groups. Histological observation of the endometrial biopsy samples from recurrent miscarriage patients gave menstrual cycle datings that ranged from day LH +2.5 to LH +6.5 with retarded endometrium (< day LH +5) in 12 of 35 (34%) patients. Of these 12 patients, 10 (83%) had low PP14 concentrations and six (50%) had low CA 125 concentrations in their uterine flushings. In the recurrent miscarriage patients with histologically normal (> or = day LH +5) endometrial development, 10 out of 23 (43%) also had low PP14 concentrations and 8 out of 23 (35%) had low CA 125 in their uterine flushings. The results suggest that PP14 is better than CA 125 as a marker for endometrial function in this group of women. In some cases (52%) the low concentrations of PP14 in the uterine flushings could be explained by retarded endometrial development but for the others the reduction in PP14 concentration in the uterine flushing was not associated with retardation of endometrial development.

Objective: To study the time course of capacitation, spontaneous, and A23187-induced acrosome reaction of human spermatozoa during 8 hours incubation in vitro using the chlortetracycline (CTC) assay with a revised fluorescent pattern classification.Design: Fertile donor spermatozoa were isolated by direct swim-up and incubated in Earle's balanced salt solution for up to 8 hours. At hourly intervals, spermatozoa were stained with CTC before and after the addition of A23187 to induce the acrosome reaction.Setting: The University Clinic, Jessop Hospital for Women, Sheffield, United Kingdom. Patients: Donors participating in the Donor Insemination Program. Main Outcome Measures: Eight fluorescent patterns identified by the CTC assay and acrosome-reacted spermatozoa detected by indirect immunofluorescence using 18.6 monoclonal antibody.Results: Using a statistical model defined by analysis of deviance allowed rationalization of the CTC pattern classification by grouping together patterns that showed a similar and significant change over time. In addition, spontaneous and A23187-induced acrosome-reacted spermatozoa identified by the CTC assay were shown to be correlated significantly to those identified by indirect immunofluorescence.Conclusion: The CTC assay using a revised pattern classification offers a more precise description of human spermatozoa capacitation in vitro. Also, CTC-identified acrosome reaction (both spontaneous and A23187 induced) was confirmed independently by indirect immunofluorescence. Fertil Steril 1995;64:150-9 Key Words: Capacitation, acrosome reaction, chlortetracycline (CTC) assay, indirect immunofluorescence, human spermatozoa, A23187. InThe chlortetracycline (CTC) assay has been reported to be a suitable method to study calciumdependent events in fertilization, such as capacitation and the acrosome reaction of spermatozoa (1). Chlortetracycline binds specifically to membrane-as- Received July 27, 1994; revised and accepted February 13, 1995. * Supported by the Infertility Research Trust, University of Sheffield, Sheffield, United Kingdom. sociated calcium, resulting in a series of fluorescent patterns on the head of sperm, which show a change over time. In nonhuman studies, CTC fluorescent patterns have been classified according to the species studied, in mouse (2), and horse (3). The CTC assay provides a method by which calcium-associated membrane changes on the head of sperm, which occur during the capacitation process, can be observed directly. Hence, the CTC assay provides an area of great interest in reproductive biology. Lee and co-workers (4) first described a classification for CTC patterns in human sperm and were able to quantify the changes in patterns during time course studies. The classification consists off our major patterns: early fresh (EF) for un capacitated sperm, shown by a bright band of fluorescence in