The routine assessment of sperm motility at room temperature and 37 degrees C.

The World Health Organization (WHO) laboratory manual (1992) states that assessment of sperm motility can be performed at either 37 degrees C or room temperature (20-24 degrees C). The motility of spermatozoa in 44 semen samples (22 fresh samples and 22 frozen-thawed samples) was assessed at both of these temperatures and a significant difference in the motility profiles was noted, specifically an increase at 37 degrees C in the percentage (expressed here as median and ranges) of spermatozoa with excellent progressive motility and an overall increase in the percentage with total progressive motility. With fresh samples the excellent progressive motility increased from 41 (19-53) to 54 (30-66) and the overall motility from 58.5 (39-74) to 65.0 (40-79). With the frozen-thawed samples the excellent motility increased from 14 (1-33) to 25 (6-45) and the overall motility from 30.5 (14-51) to 33.0 (16-52). As the WHO laboratory manual was published 'In response to a growing need for the standardisation of procedures for the examination of human spermatozoa' it is proposed that only one temperature for routine analysis should be used, namely 37 degrees C, which may have more physiological relevance and eliminate effects of fluctuations in ambient laboratory temperature.

The screening for cytomegalovirus antibody in semen donors and recipients within a donor insemination programme.

A total of 67 semen donors (62 Caucasian and five non-Caucasian) were tested for the presence of immunoglobulin G antibodies to cytomegalovirus (CMV). Ten (16%) of the Caucasian donors tested positive for CMV, while four (80%) of the non-Caucasian donors were positive. A total of 131 women receiving donor insemination (114 Caucasian and 17 non-Caucasian) were tested. Of these, 43 (38%) of the Caucasian recipients tested positive for CMV, while 16 (94%) of the non-Caucasian recipients were positive. There was a significant association between the incidence of positive tests and the age of the Caucasian recipients.

The detection of antisperm antibodies in serum: a comparison of the tray agglutination test, indirect immunobead test and indirect SpermCheck assay.

Testing for antisperm antibodies (ASAs) is an important part of the work-up of the sub-fertile couple, yet there is little consensus regarding the most appropriate methods. The SpermCheck assay (GSC; Bio-Rad Laboratories Inc., Diagnostics Division, Hercules, CA, U.S.A.) is supplied with wash buffer, controls and bead reagent which detects all three major classes of ASAs (IgA, IgG and IgM) in a single test. This study compared results on a bank of samples using the tray agglutination test (TAT), immunobead test (IBT), GSC and a modified SpermCheck assay to detect a single isotype in each test (SISC). The IBT and SISC showed excellent correlation, with 127/141 (90.1%) tests agreeing. There was an apparent lack of sensitivity to IgM with GSC as 8/15 (53.3%) samples testing positive with IBT and 7/15 (46.7%) testing positive with SISC were negative with GSC. Of the 24 IBT-negatives, seven (29.2%) were positive for TAT, indicating a high incidence of non-immunological agglutination, though this decreased as the TAT titre increased. The proportion of samples testing positive for IBT increased with TAT titre: 3/20 (15.0%) for TAT-negative samples, 6/10 (60.0%) for low titres and 21/24 (87.5%) for high titres. This was also observed when comparing the GSC with TAT. The TAT therefore appears useful as a first-line screen, whilst the inability of the GSC to adequately detect IgM limits its use as an indirect test. Both the IBT and SISC can be used to further investigate the type and class of ASA present.

Use of human zonae from cryopreserved oocytes in a test to assess the binding capacity of human spermatozoa.

Aged unfertilized oocytes from an assisted conception programme were cryopreserved and then utilized after thawing in a zona-binding assay. Oocytes frozen using dimethyl sulphoxide (DMSO) showed poor survival post-thaw (2/40, 5%) compared to those frozen with propanediol (PROH) (63/134, 47%). When the zonae were exposed to spermatozoa from fertile donors, those frozen with DMSO showed a significantly higher number of bound spermatozoa than did those frozen with PROH (P < 0.002). In both groups, oocytes which failed to survive the freeze-thaw processes had greater numbers of bound spermatozoa than did those which survived (P < 0.05). Oocytes from cases of failed fertilization showed no difference in their rate of sperm binding compared with oocytes from cases in which some fertilization had occurred. Zonae frozen in PROH but which were from oocytes which were not viable after thawing were used to assess the binding of spermatozoa from men who had failed previously to fertilize their partner's oocytes in vitro and spermatozoa from men with poor quality semen and who had elected for treatment by micro-injection sperm transfer. The number of spermatozoa bound to zonae was reduced significantly in both groups compared to a fertile donor.

Does elective cryopreservation of all embryos from women at risk of ovarian hyperstimulation syndrome reduce the incidence of the condition?

OBJECTIVES: To analyse the incidence and factors associated with the ovarian hyperstimulation syndrome (OHS) in our IVF/GIFT programme before and after the introduction of a strategy to cryopreserve all embryos from women judged to be at risk. DESIGN: Two hundred forty-one consecutive IVF/GIFT cycles from January to December 1989. SETTING: Specialist fertility unit, Manchester, UK. INTERVENTIONS: Pituitary suppression was effected by a daily subcutaneous injection of buserelin (500 micrograms) beginning 7 days before the expected menses. The ovarian stimulation was with variable amounts of human menopausal gonadotrophin. Ovulation was induced with 10,000 i.u. human chorionic gonadotrophin (hCG). From January to May (period A), gametes/embryos were replaced and 2000 i.u. hCG given, irrespective of the serum oestradiol (E2) concentration. From June to December (period B), all the embryos from women with an E2 > 3500 pg/ml on the day of ovulatory trigger were electively cryopreserved. MAIN OUTCOME MEASURES: Serum E2, features of moderate or severe OHS, clinical pregnancies. RESULTS: The OHS occurred in 10/105 (9.5%) and 12/136 (8.8%) cycles in periods A and B, respectively. Fewer women (6% versus 60%, P < 0.05) who had their embryos cryopreserved developed severe OHS compared with women with an E2 > 3500 pg/ml who became pregnant after gamete/embryo transfer in period A. The main factors associated with the development of OHS were serum E2 concentrations > 3500 pg/ml, whether gamete/embryos were replaced and the additional hCG given, the occurrence of a pregnancy and the presence of polycystic ovary disease. CONCLUSION: The elective cryopreservation of all embryos from women with high E2 levels reduced the severity, but not the incidence of symptomatic OHS.

The use of cryopreserved aged human oocytes in a test of the fertilizing capacity of human spermatozoa.

The use of cryopreserved aged human oocytes in a diagnostic test of sperm fertilizing ability was evaluated. Oocytes arising from assisted conception cycles and showing no signs of fertilization 48 h post-insemination were cryopreserved by one of two methods. An ultrarapid method using dimethyl sulphoxide gave poor post-thaw results, with only 5/69 (7.2%) oocytes surviving. Oocytes frozen by a slow method using propanediol as the cryoprotectant gave better survival rates (359/594; 60%). Fertilization by donor spermatozoa of these thawed oocytes was poor (15/63; 24%) when the zona pellucida was left intact. To improve this, the zona was enzymatically removed using pronase. These zona-free oocytes were then inseminated with spermatozoa from a fertile donor or from men previously exhibiting fertilization failure in an in-vitro fertilization treatment cycle. The fertilization rate in the patient group (41/91; 45%) was significantly lower than in the donor group (16/18; 89%) (P less than 0.02). There was also a significant (P less than 0.03) reduction in the median number of pronuclei per oocyte (2.9 versus 4.5). These results show that aged oocytes can be effectively cryopreserved to establish a bank for use in a test to identify men with impaired sperm fertilizing capacity.

Cryopreservation of human embryos at the pronucleate, early cleavage, or expanded blastocyst stages.

Supernumerary embryos following treatment by IVF or GIFT were cryopreserved at the pronucleate, early cleavage or expanded blastocyst stages. The success of embryo cryopreservation at these stages was evaluated in terms of (i) the proportion of embryos surviving the freeze/thaw procedure; (ii) the proportion of patients reaching embryo replacement; and (iii) the incidence of pregnancy per replacement. Significantly more embryos survived when frozen/thawed at the pronucleate (44/61; 72%) or early cleavage stages (48/80; 60%), than at the expanded blastocyst stage (13/34; 38%). A significantly higher proportion of patients had embryo replacements when embryos were frozen/thawed at the pronucleate (17/19; 89%) or early cleavage stages (21/24; 88%), than at the expanded blastocyst stage (9/17; 53%). Following replacement of frozen/thawed pronucleate and early cleavage stage embryos, clinical pregnancy rates of 8/17 (47%) and 3/21 (14%) clinical pregnancies were achieved, respectively. No pregnancies were achieved following replacement of frozen/thawed expanded blastocysts.

The cryopreservation of donor semen by a simplified method: use in an IVF and GIFT programme.

The cryopreservation of semen used in assisted reproduction procedures was carried out exclusively by a simplified method in which a mixture of semen and cryoprotectant was contained in 1-ml tuberculin syringes and plunged directly into liquid nitrogen. Donor semen samples halved and frozen in syringes and in straws in a controlled-rate freezer showed no significant difference in post-thaw motility (P = 0.217) or survival (P = 0.217) after 30 min. However, after 180 min the survival rate showed a significant reduction in syringes (P = 0.045). A significant difference (P less than 0.00008) in the rate of fertilization of oocytes was seen in IVF cycles using frozen-thawed donor sperm (58/142, 42%) when compared to fresh sperm from husbands (2315/3926, 59%). A significant reduction (P less than 0.00005) in fertilization rate was also observed in the case of supernumerary oocytes in GIFT cycles with the cryopreserved donor sperm (29/132, 22%) compared to the husbands' sperm (239/514, 46%). However, the pregnancy rate following IVF and embryo replacement was the same after fertilization with fresh sperm (75/351, 21%) as opposed to frozen sperm (3/14, 21%). Furthermore, a higher pregnancy rate was observed in GIFT with frozen donor sperm (9/19, 47%) than with fresh sperm from husbands (28/103, 27%), though this was not statistically significant (P = 0.079). These results show this simplified methods of semen cryopreservation to be effective when used in an IVF and GIFT programme, giving pregnancy rates comparable to fresh normospermic semen samples. The method is simple, quick and inexpensive.

Endocrine changes in women conceiving during treatment with an LHRH agonist.

We report on eight patients who conceived during pituitary desensitization with buserelin in the luteal phase of the menstrual cycle. Pregnancy was diagnosed between day 12 and 21 of buserelin administration. Analysis of serum luteinizing hormone on day 12 showed that pituitary desensitization occurred in conjunction with increasing production of ovarian steroid hormones. Serum concentrations of human chorionic gonadotrophin (HCG) were less than 10 IU/l on day 1 of buserelin administration for seven of the eight patients. The serum concentration of HCG on day 12 showed a median value of 722 IU/l (range 14.6-798 IU/l). Five of the eight patients were given HCG support (10,000 IU) following the diagnosis of pregnancy--three of these patients have ongoing pregnancies and the remaining two had blighted ova on scan. Of the remaining three patients, one had a singleton pregnancy which miscarried at 9 weeks, one had a blighted ovum on scan and bled per vagina shortly after this, and one bled per vagina prior to a scan being carried out. Our results show that pregnancy can occur during pituitary desensitization with buserelin, despite patients being counselled not to have unprotected intercourse in the cycle during which administration commences. An HCG assay on day 1 of buserelin administration is not helpful. Pregnancy should be suspected when ovarian steroid production persists despite complete pituitary down-regulation.

A quantitative assessment of somatostatin-like and neuropeptide Y-like immunostained cells in the frontal and temporal cortex of patients with Alzheimer's disease.

Immunocytochemical studies utilizing radioimmunoassay and morphological techniques have provided conflicting evidence for the involvement of somatostatin and neuropeptide Y in Alzheimer's disease (AD). However, previous investigators have not considered the effects of cortical atrophy in AD tissue on their findings. This study reports the numbers of somatostatin-like (SLI) and neuropeptide Y-like immunoreactive (NPYLI) neuronal perikarya and the length of SLI and NPYLI immunoreactive fibres, with appropriate corrections for atrophy in 6 control and 6 AD cases. There were significantly fewer SLI neurones in AD in layers II + III combined from the temporal cortex, and fewer NPYLI neurones in layers V + VI in both frontal and temporal cortices. Using a randomized method to quantify immunostained fibre length in the neuropil, an analysis of variance revealed no significant differences in the mean SLI or NPYLI fibre length per cortical strip between control and AD groups in frontal or temporal cortex. However, using a second measure of fibre length by tracing the fibres attached to consecutive immunostained perikarya, there were significant reductions in the AD brains in the mean fibre length per cell in layers V + VI for SLI in the temporal cortex, and for NPYLI in the frontal cortex. This reduction in fibre length per individual cell was presumably masked by the large variation in the fibre length found between cases using the randomized approach. It was concluded that in order to evaluate the involvement of these neuropeptides in AD from any measurements of concentration, it is essential to include some compensation for the extent of cortical atrophy that occurs with the disease.