Structure of sperm activating protein.

Serum is used as an additive in the preparation of human spermatozoa for fertilization in vitro, as it is superior to other body fluids in supporting sperm motility. We recently purified the major sperm activating macromolecule present in serum and showed it to be a complex of immunoglobulin and apolipoprotein A-I. This complex, which we named sperm activating protein (SPAP), has now been further characterized using partial proteolysis in combination with different immunological methods. SPAP was shown to interact only with antibodies against immunoglobulin G and more specifically with those against IgG4. The bacterial expression products C23 and ZZ-T (which bind to specific sites on the IgG molecule) bound in similar ways to SPAP as to IgG4 and did not hinder proteolytic cleavage of SPAP, indicating that apolipoprotein A-I is not bound closely to the binding sites of these proteins. Purified F(ab')2 fragment from SPAP was also shown to contain apolipoprotein A-I, and had a higher MW than the corresponding fragment from IgG4. Taken together, the most plausible (and in our view only possible) structure of SPAP shows an apolipoprotein A-I molecule bound in the pocket formed between the Fab arms of an IgG4 molecule. Anti-SPAP antibodies visualized by secondary fluorescein isothiocyanate (FITC)-labelled antibodies were bound to the postacrosomal part of the spermatozoa, indicating that SPAP is specifically bound to this area, and directly interacts with the spermatozoa. Based on these and earlier experiments it is speculated that SPAP acts in the lower part of the female genital tract. The benefit of SPAP should be at its greatest in these regions, and it is also possible that SPAP exerts a selection mechanism, as those spermatozoa affected by SPAP acquire increased motility, which might be important in order to reach the upper part of the female genital tract. Further exploration of the biological role of SPAP may indicate its diagnostic and therapeutic possibilities.

Serum levels of luteinizing and follicle-stimulating hormones in normal and poor-responding patients undergoing ovarian stimulation with urofollitropin after pituitary downregulation.

We were interested in the degree of downregulation in women with poor response to in vitro fertilization (IVF) treatment. Ten women who were poor-responders, arbitrarily defined as having fewer than three follicles with diameter > 14 mm on days 9 and 10 of urofollitropin therapy, were compared with ten women who were not poor-responders, in a standard IVF program. The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol after pituitary down-regulation did not differ between the two groups. The daily dose and the serum level of FSH were higher in poor-responders and there was a significant correlation between the dose of urofollitropin and the serum FSH level. There was no difference in serum LH level during ovulation stimulation between the groups. The clinical pregnancy rate was similar in the two groups although fewer embryos were transferred in the poor-response group. In conclusion, there was no difference with regard to pituitary suppression in normal-responders and poor-responders.

Separation of human spermatozoa with hyaluronic acid induces, and Percoll inhibits, the acrosome reaction.

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select) and Percoll, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll gradient. After translocation of separated spermatozoa from the Percoll solution to a culture medium, serum, Percoll or hyaluronic acid (Sperm Select) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of 45Ca2+ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of 45Ca2+ and the acrosome reaction, whilst Percoll inhibited both of these actions. Hyaluronic acid (Sperm Select) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll inhibited, influx of 45Ca2+ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select) and Percoll affect the acrosome reaction and the prerequisite for Ca2+ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.

Purification, structure and partial characterization of the major sperm activating protein complex in human serum.

Serum has long been known to enhance sperm motility, and the major factor that mediates this activation has been shown to be a macromolecule complex with a MW of about 250 kD. This macromolecule, named Sperm Activating Protein (SPAP) has now been purified by a four step protocol. Starting with 100 mL of serum 20 to 200 micrograms of purified SPAP was recovered (N = 40). Studies on the identification and structure of SPAP revealed that the macromolecule is formed by an IgG molecule to which an apolipoprotein A-I molecule is bound. Further studies on the structure of the complex was performed by partial proteolytic cleavage of SPAP by pepsin and papain and interaction with poly- and monoclonal antibodies. Based on these results the most plausible structure of SPAP places one molecule of apo A-I between the two arms of the Fab portion of an IgG4 molecule. Human spermatozoa incubated with polyclonal anti-SPAP serum and a second FITC-labelled antiserum showed fluorescence at a distinct band at the lower part of the sperm head, indicating that SPAP acts by direct interaction with the spermatozoa. The SPAP molecule shows a new mechanism mediated by the immunoglobulins, that may be very important for male fecundity.

Placental protein 14 in human in-vitro fertilization early pregnancies.

Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/l) and PP14 (micrograms/l) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful.

Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility.

Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex hormone-binding globulin, oestradiol-17 beta, progesterone and testosterone at stimulation and after embryo transfer during in-vitro fertilization.

Serum concentrations of sex hormone-binding globulin (SHBG), oestradiol-17 beta progesterone and testosterone were measured in 23 gonadotrophin-stimulated menstrual cycles and in the implantation period [days 11-19 after human chorionic gonadotrophin (HCG) injection] following in-vitro fertilization and embryo transfer. Nine cycles resulted in successful pregnancies, one pregnancy ended in spontaneous abortion (week 14) and 13 cycles were without conception. SHBG levels were significantly elevated above pretreatment values from day 3 after HCG injection onwards. A significant positive correlation was found between increments in SHBG (delta SHBG) during the luteal phase and oestradiol/testosterone ratios during the follicular and luteal phases. In the pregnant cycles a significant positive correlation was also found between delta SHBG during the implantation period and oestradiol/testosterone ratios during the luteal phase and the implantation period. Significant negative correlations were found between delta SHBG and testosterone during the luteal phase in pregnant and non-pregnant women as well as between delta SHBG during the period corresponding to implantation and testosterone during the luteal phase in non-pregnant cycles. The results may reflect a modulating action of the oestrogen/androgen balance upon SHBG levels in subjects with supraphysiological oestradiol levels, such as in stimulated cycles and in very early pregnancy.

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.

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.

Validation of radioimmunoassay for estradiol-17 beta by isotope dilution-mass spectrometry and by a test of radiochemical purity.

Isotope dilution-mass spectrometry (ID-MS) was used as a reference method to determine the concentration of estradiol-17 beta (E2) in five different plasma pools (concentrations ranging from 0.040 to 65 nmol/l). The same plasma pools were also subjected to radioimmunoassay (RIA) using five different antisera of largely varying specificity. With the best antiserum (E), a direct RIA apparently gave accurate results (i.e. results statistically indistinguishable from those obtained by ID-MS) at all levels except the lowest one (0.040 nmol/l). It was shown, however, that the apparent accuracy of this RIA to some extent could be due to a lowering effect of lipids in the serum masking a lack of specificity of the antibodies. With the least specific antiserum (A), accurate results were obtained only after chromatography. However, in the assay of the lowest concentration of E2 with this antiserum there was a significant overestimation, even after chromatography. The other three antisera (B, C, D) of average quality gave accurate results in assays of plasma diethyl ether extracts in various numbers of the plasma pools tested, depending on their intrinsic specificity. This specificity was not correlated with the cross-reaction reported for individual antisera. ID-MS is difficult to use in most laboratories. We demonstrate here that the validity of a RIA may in this case be assessed by a relatively simple method, the test of radiochemical purity (RP-test). This test is based on the measurements of specific activity (e.g. dpm/pg) in small consecutive fractions of the chromatographic zone which is usually employed for the RIA.