Leucocyte subpopulations in the seminal plasma and their effects on fertilisation rates in an IVF cycle.

Controversy exists on the role of leucocytospermia on fertilisation rates and IVF outcomes. The aim of our study was to identify the effect of leucocytes and leucocyte subpopulations on fertilisation rates in an IVF cycle. A prospective comparative study of the leucocyte subpopulations of seminal fluid of partners of women attending an IVF cycle was conducted. The samples underwent immunocytochemical staining. The monoclonal antibodies used in this study include CD3, CD4, CD8 (T Cells), CD14 (monocytes/macrophages), CD16 (granulocytes), CD20 (B Cells), CD45 (Pan Leucocytes), CD56 (natural killer cells) and CD69 (activated T and B Cells). Of 21 patients who were recruited into the study, seven were identified as poor fertilisers (<35%) and 14 were identified as good fertilisers (>60%). Data were analysed with SPSS version 14. The total leucocyte counts (CD45) between the poor and good fertilisers were not statistically significant. The macrophages and the monocytes (CD14) were significantly elevated in the good fertilisers group in comparison with the poor fertilisers (P < 0.05). We also found that T cells (CD2, CD4, CD8) and CD14 (macrophages) correlated significantly (r = 0.47, P value < 0.01) with the fertilisation rate. Our study confirms that the presence of leucocytes does not adversely affect the fertilisation rates and the outcome of an IVF cycle. However, macrophages and the monocytes (CD14) were significantly elevated in the good fertilisers group. The increased phagocytic activity in these individuals might increase their fertilising potential by removing spermatozoa with abnormal morphology.

Seminal plasma immunoglobulin concentrations in autoimmune male subfertility.

The presence of anti-sperm antibodies (ASAs) in seminal plasma is associated with infertility. They have been shown to reduce sperm motility, interfere with cervical mucus penetration and gamete interaction, and have been shown to reduce spontaneous fertilization and pregnancy rates. Although some causes can be determined, in the majority of cases the initial event causing the immune sensitisation and the reasons for the continuing antibody secretion remains unknown. Quantitative determination of total IgG, IgA and IgM within seminal plasma had not been previously reported in patients with and without specific ASAs. Semen samples from 512 men presenting with infertility were analyzed. One hundred and forty-six men (28.5%) had seminal fluid ASAs as determined by the MAR or TAT tests. The total seminal plasma IgG and IgA concentrations were significantly elevated in the ASA-positive groups compared with ASA-negative groups (IgG: 8.83 mg/100 ml vs. 7.15, P = 0.0008; and IgA: 2.88 mg/100 ml vs. 1.64, P = 0.0001). Only 19 samples showed seminal fluid IgM, and there was no difference between the ASA positive or ASA negative samples. The significance of these findings is discussed.

Soluble CD46 (membrane cofactor protein, MCP) in human reproductive tract fluids.

CD46 (membrane cofactor protein, MCP) is a cell surface complement regulatory protein which may have an additional role in human sperm-egg interaction. A soluble form (sCD46) has also been detected in a number of biological fluids, most notably seminal plasma. The present study has employed a monoclonal antibody-based ELISA to assay sCD46 in reproductive tract fluids in normal and pathological conditions. Large amounts of sCD46 were detected in seminal plasma of both fertile and infertile men (combined mean, 4859 ng/ml). Vasectomized men had lower levels (mean, 2421 ng/ml), indicating contributory sources both before and after the vas deferens ligation site. Pre-colostrum also contained relatively high quantities (mean, 445 ng/ml), whereas breast milk (mean, 117 ng/ml), peritoneal fluid (mean, 154 ng/ml) and follicular fluid (mean, 107 ng/ml), as well as uterine (mean, 208 ng/ml), umbilical (mean, 166 ng/ml) and peripheral (mean, 206 ng/ml) blood plasma, had sCD46 levels within a comparable range. Amniotic fluid had low sCD46 concentrations (mean, 22 ng/ml). In endometriosis, peritoneal fluid levels of sCD46 were significantly raised (mean, 199 mg/ml). These results indicate distinctive fluid compartmentalisation of sCD46 consistent with a biological function in human reproductive tract fluids.

Impairment of rat spermatogenesis following unilateral experimental ischemia.

When one testis in a rat is rendered ischemic, histologic damage occurs in the contralateral testis, which can be explained on immunologic grounds. An increase in the quantity of a naturally circulating cytotoxic antitestis antibody or an alteration in the specificity of this antibody was observed via cytotoxic antibody tests. Immunocytochemical techniques further demonstrated this phenomenon, showing binding of fluorescein-labeled antibody to sections of normal control testis or to sections of contralateral testis. It appeared that the ischemic testis presented altered ischemic cells or normal cells to the immune system as its blood-testis barrier was broken down. It is suggested that to leave a necrotic testis within the scrotum following prolonged torsion of the testis in the human may be a reason for subfertility or infertility in this type of condition, and a similar result may occur following unilateral testicular damage caused by varicocele or orchitis.

Peritoneal fluid concentrations of interleukin-11 in women with endometriosis.

OBJECTIVE: To determine the peritoneal fluid concentrations of interleukin-11 (IL-11) in women with endometriosis as compared with the control group. DESIGN: A prospective, controlled study. SETTING: The obstetrics and gynecology department of a teaching hospital and a university immunology department. PATIENT(S): Sixty consecutive women undergoing laparoscopic surgery for benign gynecological indications. INTERVENTION(S): Peritoneal fluid was obtained during laparoscopy, and the concentration of IL-11 was measured. MAIN OUTCOME MEASURE(S): Concentration of IL-11 in correlation with the presence of endometriosis, its stage, and the phase of the menstrual cycle. RESULT(S): IL-11 was detectable in the peritoneal fluid of 64% of women tested. Concentrations of IL-11 showed no correlation with the presence of endometriosis, the American Fertility Society stage of the disease, or the phase of the menstrual cycle. CONCLUSION(S): We found no evidence to suggest that IL-11 is involved in the pathogenesis of pelvic endometriosis.

Epidermal growth factor (urogastrone) in human seminal plasma from fertile and infertile males.

Epidermal growth factor/urogastrone (EGF/URO) was measured in the seminal plasma of 214 untreated patients attending an infertility clinic. Patients were assigned groups according to sperm density and progressive motility and in the case of azoospermia into an obstructive etiology or germinal failure. There was no significant variation in mean EGF/URO levels between groups, suggesting that this peptide plays no role in the density or motility of sperm associated with fertility. Other functions this peptide may fulfill in the female reproductive tract, including that of immunosuppression, are discussed.

Improved seminal characteristics in infertile men after a conservative treatment regimen based on the avoidance of testicular hyperthermia.

Evidence to support the contention that avoidance of testicular hyperthermia (due to hot baths and/or tight clothing) will improve fertility is, in the main, anecdotal. Semen samples derived from 128 infertile men were analyzed before and after a 3-month conservative treatment regimen. Six seminal characteristics were studied. Clinical evaluation included normal bath temperature, types of underwear worn, and past history of hernia repair, orchiopexy, or varicocelectomy. The patients were divided into three groups (A to C) according to sperm density and then subclassified on the basis of progressive motility status. The results show that a significant, or approaching significant, improvement in seminal characteristics occurs in certain groups of oligospermic individuals with pretreatment progressive motility of less than 40%. In clinical trials, it should be recognized that any improvement in seminal quality following pharmacologic or surgical intervention may include a contribution from conservative treatment.

Follicular fluid concentrations of interleukin-12 and interleukin-8 in IVF cycles.

OBJECTIVE: To investigate the role of interleukin-12 (IL-12) and IL-8 in the periovulatory follicular fluid during in vitro fertilization cycles. DESIGN: A prospective study. SETTING: Reproductive Medicine Unit, Liverpool Women's Hospital, United Kingdom. PATIENT(S): Women undergoing in vitro fertilization treatment. INTERVENTION(S): IL-8 and IL-12 concentrations in follicular fluid samples that had been collected during transvaginal oocyte retrieval were measured using an enzyme-linked immunosorbent assay (ELISA). Cytokine concentrations were correlated to fertilization rates and treatment outcome. MAIN OUTCOME MEASURE(S): Fertilization rates and ultrasonographic evidence of intrauterine pregnancy by 4 weeks after embryo transfer. RESULT(S): Failed fertilization in women with detectable IL-12 was significantly higher (45.5%) than in the IL-12 negative group (6.1%), P=.01. None of the women with detectable IL-12 achieved a pregnancy at the end of the treatment (P=.01). IL-8 was present in the follicular fluid of all women, and no difference in its concentrations was found between the pregnant and nonpregnant groups. No correlation was found between the follicular fluid concentrations of IL-8 and fertilization rates. CONCLUSION(S): The presence of IL-12 in the follicular fluid appears to be associated with a negative outcome in IVF treatment. Interleukin-8 appears to be an essential part of folliculogenesis, although its concentration is not associated with fertilization or implantation rates.

Human sperm head morphometric distribution and its influence on human fertility.

OBJECTIVE: To study the distribution of live sperm head size in semen and sperm preparations as a predictor of fertility. DESIGN: Prospective blind clinical trial. SETTING: Academic tertiary referral center. PATIENT(S): One hundred fifty-five patients undergoing IVF treatment. Females with conditions negatively influencing fertilization were excluded. INTERVENTION(S): Morphometric analysis (head area, major axis, minor axis, and elongation ratio) of video images of sperm in semen and swim-up preparations used for IVF treatment was performed with a Hamilton-Thorne analyzer V 8.1 (Hamilton-Thorn Research, Beverly, MA). MAIN OUTCOME MEASURE(S): Oocyte fertilization. RESULT(S): Seventy-four percent of patients achieved fertilization. Fertilizers and nonfertilizers had different sperm head area distribution. The fertilizers had a significantly smaller interquartile range of sperm head area and of major axis in both semen and sperm preparation compared with the nonfertilizers. A subgroup of men who had fathered a child naturally had a more uniform sperm head area in semen with a significantly smaller median compared with those who failed to father a child naturally with their healthy female partner. We used multiple logistic regression applying forward stepwise selection of variables in building three predictive models of probability of fertilization. CONCLUSION(S): Successful IVF or history of fathering a child was associated with a more uniform sperm head area in semen and sperm preparation.

Role of mitotic control in spermatogenesis.

OBJECTIVE: To study the correlation between the incidence of sex chromosome aneuploidies in the somatic cells and spermatozoa in karyotypically normal infertile men and fertile donors. DESIGN: A prospective, phase two, controlled study. SETTING: A teaching Hospital Reproductive Medicine and Medical Genetics Units. PATIENT(S): Ten patients with idiopathic oligozoospermia and 10 sperm donors with proven fertility, all with a normal karyotype 46, XY. INTERVENTION(S): Multicolor fluorescence in situ hybridization (FISH) of peripheral blood lymphocytes and spermatozoa using a probe cocktail containing the alpha satellite DXZ1 for the X centromere, DYZ1 for the heterochromatic region of the long arm of the Y, and cosmids D21S259, D21S341, and D21S342 for Down syndrome critical region of chromosome 21. MAIN OUTCOME MEASURE(S): The incidence of chromosome X, Y, and 21 aneuploidies in peripheral lymphocytes and spermatozoa in both groups. RESULT(S): The incidence of aneuploidies related to chromosomes X, Y, and 21 were significantly higher in peripheral lymphocytes and spermatozoa of infertile men compared with donors. There was a positive correlation between the incidence of chromosome aneuploidies in the somatic cells and sperm in all men. CONCLUSION(S): These findings provide suggestive evidence for the importance of mitosis in spermatogenesis and the role of mitotic instability in unexplained oligozoospermia.

Evaluation of different sperm function tests as screening methods for male fertilization potential--the value of the sperm migration test.

OBJECTIVE: To assess the value of different sperm function screening tests in predicting fertilization. DESIGN: Prospective study. SETTING: Academic tertiary referral center for fertility treatment. PATIENTS: Ninety-five couples attending for initial screening and IVF-ET. Only cycles where three or more grade I oocytes were collected were included, and patients with endometriosis were excluded. INTERVENTIONS: Each patient had a standard semen analysis, cervical mucus (CM) penetration test, hypo-osmotic swelling test, and sperm migration test performed between 4 and 8 weeks before IVF-ET. MAIN OUTCOME MEASURES: The correlation between sperm function test results and the percentage of fertilized oocytes and the power of the tests to predict fertilization. RESULTS: The sperm migration test correlated highly with fertilization rate (r = 0.62) and was most useful in identifying the group of patients likely to achieve fertilization (Odds ratio [OR] 0.07, confidence interval [CI] 0.02 to 0.2). The CM penetration test showed a moderate correlation with fertilization rate (r = 0.45) and some predictive power (OR 0.37, CI 0.13 to 1.00). Sperm concentration, but not motility or normal morphology, showed slight correlation with fertilization rate (r = 0.28) but the combination of normal semen parameters did not distinguish patients likely to achieve fertilization (OR 1.51, CI 0.62 to 3.65). The hypo-osmotic swelling test did not correlate with fertilization rate (r = 0.21). CONCLUSIONS: This study evaluated the predictive power of several simple tests available for use in most laboratories as screening tests of sperm fertilization potential. Apart from sperm concentration, normal traditional semen characteristics were of little clinical benefit. The hypo-osmotic swelling test had no predictive power. The CM penetration test correlated with fertilization rate but might be difficult to perform routinely as a continuous supply of suitable CM would be required. The sperm migration test proved to be the best discriminator of sperm fertilization potential and should be considered as a first level screening test in the assessment of male fertility.

Effect of abnormal hypo-osmotic swelling test on fertilization rate and pregnancy outcome in in vitro fertilization cycles.

OBJECTIVE: To assess the value of the hypo-osmotic swelling test in predicting fertilization, pregnancy, implantation, miscarriage, and live birth rates in IVF-ET cycles. DESIGN: Prospective study. SETTING: Academic tertiary referral center for fertility treatment. PATIENTS: Three hundred twenty-six couples having IVF-ET for tubal damage or male factor infertility with the female partner < 38 years of age. INTERVENTIONS: Each male had a hypo-osmotic swelling test performed between 4 and 8 weeks before IVF-ET. MAIN OUTCOME MEASURES: Fertilization, implantation, miscarriage, and live birth rates. RESULTS: Eighty of 326 men had abnormal hypo-osmotic swelling tests. An abnormal test was not associated with lower fertilization rates (odds ratio [OR] = 1.14; 95% confidence interval [CI] = 0.97 to 1.14) or pregnancy rate (OR = 0.98; CI = 0.50 to 1.96). However, although couples with a normal test had a miscarriage rate of 26.9% (14/52), in the group with an abnormal test the miscarriage rate was 50.0% (7/14) (OR = 0.37; CI = 0.09 to 1.49). This resulted in a reduction in the live birth rate from 14.1% in the group with a normal test to 11.8% in patients with an abnormal test (OR = 1.23; CI = 0.45 to 3.87). CONCLUSIONS: The hypo-osmotic swelling test has little value in predicting fertilization in IVF-ET procedures. However, an abnormal test may help predict adverse outcome if pregnancy is achieved.

Evaluation of the role of mitotic instability in karyotypically normal men with oligozoospermia.

OBJECTIVE: To estimate the incidence of sex chromosome aneuploidies in the somatic cells of karyotypically normal infertile men and fertile donors. DESIGN: A prospective, two-phase, controlled study. SETTING: Reproductive medicine and medical genetics units of a teaching hospital. PATIENT(S): Ten patients with oligozoospermia and 10 sperm donors with proved fertility, all with a normal karyotype 46 XY. INTERVENTION(S): Multicolor fluorescence in situ hybridization (FISH) of peripheral blood lymphocytes using a probe cocktail containing the alpha satellites DXZ1 for the X centromere and DYZ1 for the heterochromatic region of the long arm of the Y and the radiolabeled alpha satellite D18Z1 for chromosome 18. MAIN OUTCOME MEASURE(S): The incidence of sex chromosome aneuploidies in both groups. RESULT(S): A 13-fold increase in sex chromosome aneuploidies was observed in the somatic cells of infertile men with "unexplained" oligozoospermia compared to donors (P=.008). CONCLUSION(S): These findings provide suggestive evidence for the existence of an inherent mitotic instability in men with unexplained oligozoospermia.

The hypo-osmotic swelling test in recurrent miscarriage.

OBJECTIVE: To determine whether there is any association between sperm membrane integrity as determined by the hypo-osmotic swelling test score and unexplained recurrent miscarriage. DESIGN: Prospective observational study. SETTING: Tertiary referral center for recurrent miscarriage. PATIENT(S): Semen samples from 20 male partners of women who had had three or more first trimester miscarriages of unexplained etiology and semen samples from 20 prospective semen donors of unknown fertility potential. MAIN OUTCOME MEASURE(S): Sperm density, sperm motility, sperm morphology, and hypoosmotic swelling test score. RESULT(S): There was no difference in the median sperm density, the mean sperm motility, or the mean sperm morphology between the two groups. However, the recurrent miscarriage group had a significantly lower hypo-osmotic swelling test score than the control group. CONCLUSION(S): The hypo-osmotic swelling test score is significantly lower in samples from men whose partners have had unexplained recurrent spontaneous abortions. With the exception of cytogenetic abnormalities in peripheral blood karyotype, this is the first study to identify a male factor component in recurrent miscarriage.

Seminal plasma lactoferrin concentrations in normal and abnormal semen samples.

Although the iron-chelating protein lactoferrin is secreted by the seminal vesicles, the precise role of lactoferrin in semen is unclear. This study aimed to determine whether there is any association between seminal lactoferrin concentrations and normal and abnormal semen samples with and without leucocytospermia. Lactoferrin concentrations were measured by radial immunodiffusion of semen samples from 368 men attending a regional andrology referral center. Routine seminal analysis, including the presence of leucocytospermia, was also performed. Results showed increased seminal lactoferrin in samples showing oligospermia (13.3 mg/100 ml) and oligoasthenospermia (13.4 mg/100 ml) compared to normospermic samples (11.2 mg/100 ml). There were no significant differences in seminal lactoferrin between normospermic samples and azoospermic samples or asthenospermic samples with normal sperm density. Although there was a trend toward increased lactoferrin concentration with leucocytospermia, this was not significant. Possible causes for raised lactoferrin in association with oligospermia are discussed.

The incidence and significance of testicular microlithiasis in a subfertile population.

The objective of this study was to assess the frequency of testicular microlithiasis (TM) in infertile men who underwent testicular ultrasound and to determine any causative or associated factors. The case notes of 159 consecutive patients who were referred for testicular ultrasound in the investigation of male factor infertility were reviewed. Microcalcification was found in 10 cases (6.2%). This was unilateral in all cases and six patients had clinical evidence of a varicocele. Five cases had minimal calcification and five had marked TM. On patient had a past history of testicular maldescent and another of testicular torsion. Sperm function (as assessed by sperm count, motility and the sperm migration test) was variable within the 10 patients and there was no correlation with hormone profiles or testicular size. There was a statistical difference between a number of investigations in those patients with minimal degrees of calcification and those with TM (sperm migration test (SMT), namely sperm migration and sperm motility (p < 0.05, Mann-Whitney U test)). The results showed a higher than expected incidence of TM. Patients with marked TM seemed to have poorer sperm function than those with minimal calcification. There was a high incidence of co-existing pathology, for instance scrotal varicocele and cryptorchidism, although the numbers in this study were small and further studies need to be carried out to determine the exact nature and significance of this condition.

A simplified system of human milk banking.

A simplified system of human milk banking, from milk supplied from home or hospital, has been evaluated for use in a neonatal intensive care unit. Twenty milk samples were obtained at a single expression using a standard hand pump and divided into three parts. Analyses were performed on the raw milk and on samples stored at -20 degrees C for 1 week and 1 month. No pathogens were isolated from any samples and the counts of Staphylococcus albus in the raw milk remained unchanged after storage. 19% of the cells in the original milk survived freezing and remained viable. There was a loss of bacteriostatic activity after storage for one month but significantly less than that caused by pasteurization. No change in levels of IgA, IgM, IgF, lactoferrin, lysozyme, C3 and C4 was apparent and concentrations of amino acids and fatty acids also remained unchanged after storage. We conclude that milk can be safely and conveniently stored by this method without loss or damage to the components of raw breast milk important for preterm and sick infants.

Second to fourth digit ratio (2D:4D) and testosterone in men.

AIM: To investigate the relationship between 2D:4D and testosterone in men attending an infertility clinic and men drawn from the general population. METHODS: Data on 2D:4D and testosterone from two samples were collected: (1) 43 men attending an infertility clinic, and (2) 51 men drawn from the general population without regard to fertility. RESULTS: In sample (1) there were negative associations between 2D:4D and testicular function, and men with lower 2D:4D in their right compared to left hand had higher testosterone levels than men with higher 2D:4D in their right compared to left hand. Sample (2) showed no significant associations between 2D:4D or side differences in 2D:4D and testosterone. CONCLUSION: Adult levels of testosterone may be related to aspects of 2D:4D in samples which contain men with compromised testicular function, but not in men from normative samples. Associations between 2D:4D and fertility-associated traits probably arise from early organisational effects of testosterone rather than from activational effects of current testosterone.

The evaluation of the sperm migration test as a predictor for success with intrauterine insemination.

OBJECTIVE: The objective of this study was to prospectively evaluate the sperm migration test (SMT) as a discriminator in couples undergoing intrauterine insemination (IUI). PATIENTS AND METHODS: 261 couples underwent 797 IUI treatment cycles involving gonadotropin stimulation in the three year period. All had a diagnosis of unexplained infertility. All male partners underwent a repeat standard seminal analysis and SMT prior to the female partner undergoing controlled ovarian stimulation. RESULTS: Despite apparently normal seminal analyses before referral, in 22 samples the sperm concentration, motility or morphology were abnormal (WHO criteria). Of these, 20 couples underwent 109 cycles and achieved 2 pregnancies giving a pregnancy rate of 1.8% per cycle and a cumulative pregnancy rate of 10% per couple. From the remaining couples with normal seminal analyses, 71 had an SMT <5 million/mL and 168 had an SMT >5 million/mL. The suboptimal SMT group underwent 276 cycles (3.89 cycles per couple) and achieved 18 pregnancies giving a pregnancy rate of 6.5% per cycle and a cumulative pregnancy rate of 25.4%. The normal SMT group underwent 412 cycles (2.45 cycles per couple) and achieved 60 pregnancies giving a pregnancy rate of 14.6% per cycle and a cumulative pregnancy rate of 35.7%. CONCLUSIONS: We confirm that abnormal seminal analysis leads to poor pregnancy rates with IUI. However, an SMT <5 million/mL despite normal seminal analysis (WHO criteria) also leads to significantly worse pregnancy rates. We would recommend that prior to IUI, couples are screened using the SMT.