Assisted ejaculation combined with in vitro fertilisation: an effective technique treating male infertility due to spinal cord injury.

Infertility due to spinal cord injury (SCI) in males has been identified for decades as an area of major concern and techniques for assisted ejaculation are available. There has not been an overall consensus regarding which type of assisted procreation is the most appropriate for these couples. We describe here our experience from a programme based on assisted ejaculation combined with in vitro fertilization (IVF). Twelve couples have been treated so far and altogether 22 cycles with ovum pick-up have been completed. Fertilisation of the oocytes was obtained in 18 of these cycles. The overall oocyte fertilisation rate was 49%. Embryo transfer took place in 17 cycles, leading to seven clinical pregnancies. Four of the pregnancies are delivered or are ongoing, whereas three ended in first trimester spontaneous abortion. Thus our initial experience suggests that assisted ejaculation in combination with IVF is an effective option for these couples.

Sperm count and motility influence the results of human fertilization in vitro.

In order to select sperm characteristics that can predict the outcome of in-vitro fertilization-embryo transfer (IVF-ET), semen samples delivered in conjunction with this treatment were studied carefully. We have analysed these data retrospectively in relation to the outcome of treatment. Ninety-one couples were treated for tubal infertility by IVF-ET. Fifteen women became pregnant. Sperm were isolated from semen using a swim-up technique and in most cases 40-80 x 10(3) (range 20-120 x 10(3)) motile sperm per ovum were used for insemination. The couples were divided into three groups: group A who achieved pregnancies, group B who achieved cleaved ova but no pregnancies, and group C who achieved no ova that were cleaved 48 h after oocyte recovery. Comparisons between these groups showed that some characteristics of the native semen samples and the swim-up preparations were significantly different: the sperm concentration (P = 0.001) and total sperm count (P = 0.003) in the native sample, the number of sperm recovered during 30 min of swim-up (P = 0.001), and the specific progressive motility of sperm in the swim-up preparation, both at the time of insemination and on each day, up to 5 days thereafter (P = 0.002-0.028). No pregnancy was achieved with a sperm concentration below 26 x 10(6) ml-1 in the native sample. Some of the sperm characteristics studied in this paper may be of value in the pretreatment evaluation for IVF treatment.

Comparison between a conventional index of progressive sperm motility and movement variables analysed by CellSoft.

Two different techniques of human sperm motion analysis were compared. In 25 ejaculates, diluted to an appropriate concentration, the sperm motion index called "specific progressive motility" (SPM) was assessed by a modified conventional method. The same sperm preparations were also analysed by the CellSoft system for computer assisted sperm analysis (CASA). The SPM-value was found to correspond mainly to the variables motility and curvilinear velocity presented by CASA (multiple r = 0.92, P less than 0.001). Linearity of sperm progression, lateral head displacement, and beat cross frequency did not significantly contribute to the relationship between SPM and CASA. For comparative studies between SPM and CASA the logarithmic values of SPM are recommended.

Changes in human sperm chromatin stability during preparation for in-vitro fertilization.

This study was designed to define the effects of sperm preparation on sperm chromatin stability in relation to in-vitro fertilization (IVF). Semen samples used for IVF-embryo transfer (ET) in the treatment of infertility due to tubal factors were studied. Cases with semen variables below reference limits in previous samples were excluded. Sperm were prepared by a swim-up technique employing either of two different tissue culture media, Ham's F-10 or Earle's balanced salt solution. Sperm chromatin stability was tested by exposure both to sodium dodecyl sulphate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS-EDTA). Sperm head swell scores were defined under different experimental conditions and the relationship to sperm motility, morphology, fertilization rate and pregnancy occurrence was tested. No differences were seen between the chromatin stability of sperm from the original sample and that after swim-up preparation, neither immediately after completion of the swim-up procedure, nor at the time of insemination of ova. With time, the chromatin became more stable, which occurred to a similar extent both in the original sample and in swim-up preparations using Ham's F-10. Otherwise, sperm chromatin stability was unaffected by either of the two media used for swim-up. At higher incubation temperatures, decondensation in SDS was enhanced. Altogether, no correlation was found between sperm chromatin stability or enhancement of decondensation by temperature and the success of IVF treatment expressed in fertilization rates or pregnancies. The results are reassuring in that only small changes in sperm chromatin stability occurred during the preparation for IVF. As long as semen of presumably good quality is used, these changes in chromatin stability do not seem to be of clinical importance.

Morphology of seminal and swim-up spermatozoa and the outcome of in vitro fertilization and embryo transfer.

Tubal infertility was treated by in vitro fertilization-embryo transfer (IVF-ET) in 112 couples. Twenty-eight pregnancies were obtained in 140 treatment cycles. Couples are accepted for treatment in our IVF-ET programme if previous semen samples fulfil the inclusion criteria: ejaculate volume greater than 1.5 ml, concentration of spermatozoa greater than 15 x 10(6) ml-1, greater than 40% motile spermatozoa, and greater than 25% spermatozoa with normal morphology. In order to determine to which extent IVF-ET treatment results are influenced by sperm morphology, within this selected group of patients, we have retrospectively analysed the data from both original semen samples and swim-up preparations. The sperm morphology was not related to the outcome of treatment in terms of fertilization (ovum cleavage rate), early embryo development, or pregnancy. Nor was any relationship detected between early embryo development or pregnancy and the degree of improvement in morphology resulting from the swim-up procedure. However, if improvement in morphology by swim-up was high, ovum cleavage rate was low. Sperm morphology within the limits set by our inclusion criteria could not predict the outcome of IVF-ET treatment. It is further concluded that the presence of abnormal spermatozoa at the site of fertilization may be without harm if only the number of normal sperms is high enough.

The early luteal phase in successful and unsuccessful implantation after IVF-ET.

The hormonal milieu at embryo implantation after in-vitro fertilization was investigated. Superstimulation was accomplished with clomiphene citrate and human menopausal gonadotrophin (HMG) injections followed by ovulation induction with human chorionic gonadotrophin (HCG). Venous blood samples were drawn on days 2 and 8, the day of oocyte recovery being day 0. Fifteen women with successful implantation, defined as an ultrasound-verified pregnancy, were compared to 42 women with unsuccessful implantation, using a three-way analysis of variance. Oestradiol, progesterone, testosterone and sex hormone binding globulin (SHBG) did not differ between the two groups. However, the ratios of oestradiol/progesterone and of testosterone/SHBG were significantly higher in the non-fertile cycles, both on day 2 and on day 8 (P less than 0.05). Furthermore, there was a highly significant decrease in oestradiol, progesterone and testosterone between days 2 and 8 in fertile as well as in non-fertile cycles (P less than 0.001) and a highly significant increase in SHBG from day 2 to day 8 in both groups (P less than 0.001). The higher testosterone/SHBG ratio in the non-pregnant women implies a relative hyperandrogenicity in this group that might have adversely affected the uterine receptivity.

Serum factors stimulate the motility of human spermatozoa.

Motile human sperm were collected from a Percoll gradient and the effects on sperm motility of human serum, various serum fractions, follicular fluid and seminal plasma were assessed. In culture medium alone (RPMI-1640) sperm motility was lost after about 5 h. The addition of male blood serum both enhanced sperm motility and prolonged viability very significantly. Albumin, seminal plasma and follicular fluid all stimulated sperm motility but to a much lesser extent than did blood serum. No difference was noted between male serum or female serum which had been collected during the follicular or luteal phases of hormone-stimulated cycles and which contained high levels of oestradiol. Serum fractions obtained by separation on Sephacryl S-300 column were tested for their ability to enhance sperm motility. The most pronounced effect, much superior to that achieved by the albumin fraction, was obtained by a fraction with a molecular weight of around 200 kD. In conclusion, certain factors in human serum, which are different from albumin, strongly support sperm motility. The high serum concentrations of oestradiol resulting from hormone stimulation for in-vitro fertilization do not invalidate the use of serum from the same patient during sperm preparation, or in the medium used for ovum insemination and culture.

Scheduled intermittent periods of in vitro fertilization treatments.

Organization and results of an in vitro fertilization program at the Huddinge University Hospital are given from its beginning in August 1985: 6 months in advance a scheme is scheduled with 2 weeks open for treatment followed by free intervals of 3-4 weeks. Follicular development is stimulated with clomiphene citrate and hMG, and assessed by analyses of estradiol and LH in serum combined with ultrasound examinations. Following the administration of hCG, eggs are collected by transvesical aspiration guided by ultrasound. The ova are inseminated with about 50,000 motile spermatozoa, and cultured for 48 h. Up to four eggs are transferred transcervically to the uterine cavity. 158 egg pickups have been performed (August 1985 to December 1987) in 106 patients resulting in fourteen intrauterine and two ectopic pregnancies, biochemical pregnancies not counted. This protocol has restricted routine work load allowing these treatments to be part of the clinical routine. It has also allowed the application of research programs and thus optimized limited resources.

Comparison between a swim-up and a Percoll gradient technique for the separation of human spermatozoa.

Two methods of separating human sperm were compared using twenty-two semen samples. The sperm were separated by a swim-up technique or by self-migration on a Percoll gradient followed by medium change. After separation, the sperm obtained were assessed for progressive motility, ATP content, energy charge index ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) and morphology. In general, and especially for semen samples containing less than 20 X 10(6) sperm/ml, separation by Percoll gradient selected sperm that were superior to those separated by the swim-up technique. The relatively high energy charge index (greater than 0.8) showed that the sperm tolerated the separation conditions well. It is suggested that self-migration on a Percoll gradient should prove useful for obtaining sperm of high quality.

On the use of adenosine triphosphate for estimation of motility and energy status in human spermatozoa.

Following the development of a reliable technique for analysis of total ATP in semen, the ATP concentration of untreated human ejaculates was compared with progressive sperm motility during 3 continuous days following ejaculation. As correlation was poor between these variables, spermatozoa were separated by self-migration on Percoll gradients and washed free of Percoll solution. ATP concentration and sperm motility was then assessed. Aliquots of separated sperms were transferred to separate tubes, and increasing concentrations of sera were added prior to incubation at 37 degrees C for 4 days. Under these conditions a good correlation between specific progressive motility and specific ATP content was found (r = 0.88). A high concentration of human blood serum (10% v/v) was found to be necessary for maintainance of motility, but neither the addition of bovine serum albumin nor an increase in the glucose concentration had any effect on sperm motility or on the ATP concentration. Addition of different fractions of human blood serum indicated that the components mainly responsible for maintaining sperm motility are greater than 10,000 daltons. Our findings strongly indicate that ATP assessment can be used to estimate the energetic condition of a human sperm population after its separation from seminal plasma. It is suggested that this in vitro system can be used to define optimal conditions for sperm function.

Increase in progressive motility and improved morphology of human spermatozoa following their migration through Percoll gradients.

Human spermatozoa were separated on the basis of their motility in a discontinuous Percoll-gradient made up in tissue culture medium containing 10% (v/v) human serum (TCMS). Portions of ejaculates were placed on top of the gradients. After 3 h at 37 degrees C the bottom 1.5 ml was collected and the sperm washed free of the Percoll solution by centrifugation at 240 X g after dilution in TCMS. In this way the spermatozoa were separated from seminal fluid by means of the swimming rate of the sperm. When semen samples from normal men were used, total recovery of sperm after separated on a Percoll gradient was 21 +/- 2.3%. The progressive motility index increased by a factor of 15 +/- 1 when comparing separated samples with the same unseparated ejaculate, and the frequency of sperm with normal morphology increased from 60 to 85%. The improvements in these semen samples was attributable to the Percoll separation as the washing procedure itself was without effect. Using this method sperm of relatively unifirm motility and morphology can be collected. These may then be used for further biochemical and physiological studies. Improved sperm quality was also obtained when samples from patients with abnormal semen profiles were separated in this way, although the degree of improvement was much more variable than that obtained with semen from normal fertile men. This indicates that this method can be used in clinical practice in selected cases for the preparation of sperm for insemination or for in-vitro fertilization.

Surgical correction of female infertility. An analysis of results and failures in 101 cases.

During the 1970s, surgery undertaken to remedy infertility at Sabbatsberg Hospital in Stockholm involved macrosurgical techniques. The patients described here had the same pathology on either side. Sixty-three underwent different procedures for the correction of tubal pathology at laparotomy, while 20 patients had lysis of adhesions performed at laparoscopy. A further 18 patients were operated on for ovarian endometriosis. The results in terms of conceptions (ranging from 43 to 60%, depending upon the type of operation) were similar to those reported from microsurgery, but the incidence of ectopic pregnancy was higher. Second-look laparoscopy revealed the presence of adhesions and tubal block in a significant number of patients, though less often in those who ultimatively conceived. There was no difference in semen findings between patients who conceived and those who did not. Postoperative adhesions are responsible for many surgical failures, but refined methods of surgery may reduce the risk of future ectopic gestation. Only a limited number of cases remain where persistence of infertility may be due to minor anatomical or functional defects not amenable to present-day clinical evaluation.

Successful tubal reimplantation after rupture of the uterus.

A 29-year-old woman with primary infertility caused by bilateral isthmical tubal block was operated upon with bilateral cornual resection and tubal implantation. After one uncomplicated childbirth a second fullterm pregnancy ended in fetal catastrophe: the uterus ruptured at both sites of tubal implantation and a stillborn fetus was delivered by emergency cesarean section. One oviduct was detached from the uterus and the other one had to be removed. However, the maternal condition never became critical. Later on, a second tubal reimplantation was performed on the remaining side, and after a first-trimester spontaneous abortion a second healthy infant was born at elective cesarean section.

Serum levels of 4-androstene-3,17-dione in menstruating and postmenopausal women. Evaluation of a radioimmunoassay and correlation with bone mineral content and endometrial pathology.

A radioimmunoassay for serum 4-androstene-3,17-dione (A-4) using anti-4-androstene-3, 17-dione-11 alpha-hemisuccinate bovine serum albumin was applied to serum samples from 16 healthy, normally menstruating women in different phases of the menstrual cycle, from 36 healthy postmenopausal women and from 23 postmenopausal women with various bone mineral contents and endometrial conditions, undergoing an acute ACTH stimulation test. The values obtained in the normal subjects were consistent with those found in the literature. A-4 levels were significantly higher at midcycle than in the follicular phase and were significantly lower in postmenopausal than in menstruating women. ACTH increased the levels of A-4 significantly. However, no significant correlation was found between basal levels or the ACTH-induced increments of A-4 on the one hand and bone mineral content, endometrial condition and the patients' weight, age, or Broca's index on the other. This is in contrast to our previous finding of significant correlations between basal dehydroepiandrosterone sulfate and ACTH-induced increments in dehydroepiandrosterone on the one hand and bone mineral content on the other in the same clinical material. We discuss whether the differences in correlations to bone mineral content between the two adrenocortical androgens may be due to a rather more complex origin of the steroid moiety of A-4.

Peripheral hormone levels and the endometrial condition in postmenopausal women.

Thirteen postmenopausal women with benign endometrial changes including proliferative, secretory and polypous endometrium, endometrial hyperplasia and atypia (group I) and 13 randomly selected age-matched controls with normal atrophic endometrium (group II) were studied with respect to serum levels of dehydroepiandrosterone (DHA) and its sulfate (DHAS), testosterone, total estrone, estradiol-17 beta, progesterone, FSH and prolactin. Serum levels of DHA, DHAS, testosterone and total estrone were significantly higher in group I than in group II; otherwise no significant differences were found. Mean values for body weight and for Broca's index, respectively, were almost identical in the two groups. It is speculated that the adrenal androgens may affect the endometrium in two ways, viz. via peripheral conversion to estrogens and/or via direct interaction with endometrial steroid receptors. The results give further support to the hypothesis of an association between adrenocortical hyperactivity and endometrial abnormalities including endometrial carcinoma.

Adrenocortical steroids, bone mineral content and endometrial condition in post-menopausal women.

In 23 post-menopausal women, serum levels of cortisol, unconjugated dehydroepiandrosterone (DHA), dehydroepiandrosterone sulphate (DHAS), testosterone, unconjugated and total oestrone and prolactin were measured before and during an ACTH test. Significant positive correlations were found between basal levels of DHA and DHAS; DHA and unconjugated oestrone; DHA and total oestrone; testosterone and total oestrone and between unconjugated and total oestrone. ACTH significantly raised the levels of the steroids but not of prolactin. Significant positive correlations were found between basal levels and ACTH induced increments in DHA; between basal DHAS and increments in DHA and between increments in DHA and DHAS. A significant negative correlation was found between basal levels and increments in cortisol. No significant correlations were found between other combinations of hormone basal levels and/or increments. Significant positive correlations were found between basal levels of DHAS and the DHA response to ACTH respectively, and trabecular bone mineral content of the distal forearm. A significant correlation was also found between bone mineral content and pre-cancerous/cancerous state of the uterine epithelium. The results are a further support to the concept of a link between adrenal androgens and bone mineral density, and do also indicate a relation to endometrial pathology. The lack of correlation between cortisol and other steroids indicate different regulatory mechanisms. Prolactin does not seem to be involved in the regulation of the adrenal androgen synthesis.