Andrology: Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria sperm morphology

This study evaluated 120 couples undergoing in-vitro fertilizationtreatment to determine which semen parameter(s) predicted fertilizationand whether there was any consistent relationship between strictcriteria and standard assessment of sperm morphology. Strictcriteria morphology was the only significant predictor of fertilization(P = 0.0006, r² = 0.09), with a sensitivity of 94% and a specificityof 40%. A 12% cut-off point presented a negative predictivevalue of 98% and a positive predictive value of 22%. The probabilityof satisfactory fertilization is 40% with morphology <4%,which increases to 97% with normal morphology (12%). The receiveroperating characteristic curve deviated significantly from thediagonal with a 76% area wider the curve, making this a superiorpredictive test. This was augmented by likelihood ratios (LR)of 8.25 (LR +) for results with <4% normal morphology and0.15 (LR-) for results with 12% normal morphology by strictcriteria. While there was some correlation between strict criteriaand standard assessment of morphology (r²=0.35), the formerexplained only 12% (r²=0.12) of the variability in the latter.This study concludes that strict criteria morphology predictsfertilization, while other semen parameters do not. A 12% cut-offpoint makes strict criteria morphology an excellent predictorof satisfactory fertilization, while a value <4% is a goodpredictor of poor fertilization.     

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: immunolocalization of inhibin {alpha} and {beta}a subunits

Transforming growth factor (TGF) is known to have the abilityto modify mitogenic responses of tissues to other peptide growthfactors and therefore may contribute to the rapid growth rateof an embryo. Throughout the TGF superfamily there is a similarfundamental molecular architecture. Included in this superfamilyare inhibin A, activin A and activin B. It has been shown thatactivin is a powerful mesodermal inducing factor in the earlyembryo. The human embryo has shown localization of inhibin inthe gonads after 16 weeks gestation but it has not been previouslyidentified in earlier embryos. The inhibin-activin protein wasfound in a range of tissues including the liver stages 19-21() and stages 19-22 (ß); oesophagus stages 19-22 (and ß); stomach stages 21 and 22 ( and ß);gut stages 16-22 () and 21 and 22 (ß); pericardiumstages 12-22 ( and ß); gonad stages 21 and 22 (ß)stage 22 (); adrenal stages 19-22 ( and ß); urogenitalsystem states 21 and 22 ( and ß); yolk sac stage 12( and ß); mesenchyme stages 16-22 (); surface ectodermstages 13-22 () and stages 16-22 (ßa); notochord stages13-22 (ß) and stages 21 and 22 (); nasal, tracheaand bronchi stages 19-22 ( and ß) leading to speculationof the role of both subunits.     

Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding

BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) isa common cause of failure of fertilization in vitro. This studywas to determine if DSZPB is caused by defective pathways upstreamof protein kinase A (PKA) and C (PKC), or reduced protein tyrosinephosphorylation (TP). METHODS: Infertile men with DSZPB and either normal sperm morphology(NSM) 14% (n = 15) or 5% (n = 15) were studied. Sperm–ZPbinding test was performed by incubation of motile sperm withoocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP,PKA activator) or phorbol myristate acetate (PMA, PKC activator).TP of capacitated sperm in medium was assessed by immunofluorescencewith an anti-phosphotyrosine monoclonal antibody. RESULTS: For normal sperm with normal sperm–ZP binding, both PMAand dbcAMP significantly enhanced sperm–ZP binding ina dose–response manner. Only dbcAMP, but not PMA, significantlyincreased TP of capacitated sperm. In DSZPB men with severeteratozoospermia (NSM 5%), neither PMA nor dbcAMP enhancedsperm–ZP binding, despite dbcAMP significantly increasingthe TP of capacitated sperm for all samples. In contrast, forDSZPB with NSM 14%, PMA caused significantly increased spermbinding up to normal levels (40 sperm bound/ZP) in five men,and dbcAMP had a similar result in two men. Again TP was significantlyenhanced only by dbcAMP, but not by PMA. CONCLUSIONS: There is defective signalling in pathways upstream of PKC andPKA in some men with DSZPB and normal semen analysis. Stimulationof TP by dbcAMP does not enhance sperm–ZP binding capacityin DSZPB men with low TP, regardless of sperm morphology.     

Morphological and molecular characteristics of living human fetuses between Carnegi stages 7 and 23: localization of inhibin mRNA {alpha} and {beta}a subunits by in-situ hybridization

Localization of the mRNA and ßa subunits of inhibinhas previously been reported in the human gonads during thesecond trimester. Adrenal inhibin has also been reported inthe second trimester for the ßa and ßbsubunits. Investigations showing localization by in-situ hybridizationduring the first trimester have not been reported. The resultshave shown hybridization of the and ßa subunits,throughout the period of development studied, in a variety oftissues including the dorsal and thoracic aortas and pericardiumstages 13-22 (ßa subunit); liver stages 19-21 (ßa)and stages 21-22 (); mesonephros stages 21 and 22 (ßa);gonad stages ( and ßa); adrenal stages 19-22 (); surfaceectoderm stages 16-22 (ßa); mesenchyme stages 16-22(ßa); amnion stages 13-16 (ßa); yolk sacstage 12 ( and ßa); cartilage stages 19-22 (ßa);and nasal proliferation stages 21 and 22 (ßa). Whencompared with distribution of the protein subunits it was notedthat more immunostaining activity was found, suggesting thatprobes were not sufficiently sensitive enough to detect alllevels of mRNA expressed. It can be surmised, therefore, thatthe lack of visual hybridization of the mRNA cannot precludethe possibility that it is not being translated within the tissueeven though hybridization was not apparent.     

Andrology: Relationship between stimulated hyperactivated motility of human spermatozoa and pregnancy rate in donor insemination: a preliminary report

The proportion of spermatozoa exhibiting the vigorous motilitybehaviour termed ’hyperactivation‘ (HA) has beenshown to be increased following removal of seminal plasma andstimulation with chemical agents such as pentoxifylline. Theaim of this study was to examine the relationship between theproportion of HA in cryopreserved semen samples from sperm donorsand the corresponding pregnancy rates achieved by donor insemination.Cryopreserved samples from 20 men were incubated in the presenceor absence of 3 mM pentoxifylline for 1 h and the %HA determinedin each sample. The relationship between pregnancy rate, theproportion of HA spermatozoa in control and pentoxifyllinetreatedgroups and the change in %HA following pentoxifylline treatment(HA) as well as the mean semen characteristics for each donor[sperm count, motility (%), motility index, normal morphology(%), post-thaw motility (%) and post-thaw motility index] wereexamined by logistic regression of the occurrence of clinicalpregnancy with each insemination. Both HA and mean post-thawmotility index were significantly related to pregnancy ratesand together accounted for 64% of the observed variation inpregnancy rates.     

Sperm functional changes and fertilization in vitro in co-culture with human skin fibroblasts

This study was undertaken to evaluate the effects of human skinfibroblast monolayers on human sperm function and fertilizationin vitro. Sperm function was evaluated using the hamster oocytepenetration assay (HOPA) and zona binding assay (ZBA) in mediumalone and in co-culture with human skin fibroblast monolayersand suspensions. The ZBA was also studied in fibroblast conditionedmedium and in bovine oviduct cell monolayers and suspensions.Fertilization was measured both in in-vitro fertilization (IVF)couples with a normal semen analysis (first study; randomized)and in IVF couples with subnormal semen analysis (second study;each patient served as its own control). The HOPA results werenot significantly different with or without fibroblasts. Inall co-culture situations and in conditioned medium the ZBAscored significantly lower than medium alone. No significantdifferences with respect to IVF were observed between the co-cultureand the control group in either study. The mean fertilizationrate per patient was 60% in the group with normal semen analysisand 25% in the group with abnormal semen analysis. From thisstudy we conclude that although co-culture with human skin fibroblastsand epithelial cells influences the results of some sperm functiontests, it does not influence fertilization in vitro.     

Immunology: Autoimmunity to spermatozoa, asymptomatic Chlamydia trachomatis genital tract infection and {gamma}{delta} T lymphocytes in seminal fluid from the male partners of couples with unexplained infertility

The relationship between an undetected, asymptomatic Chlamydiatrachomatis genital tract infection, the concentration of andb T cells in semen and sperm autoimmunity was examined in 48male partners of couples with unexplained infertility. ImmunoglobulinA (IgA) antibodies to C.trachomatis were detected in seminalfluids from 14 (29.2%) of the men. Only four of these were positivefor circulating anti-chlamydial IgA, suggesting that the stimulusfor antibody production was within the genital tract. In contrast,four men were positive for anti-chlamydial IgG in their semen;all were also seropositive for anti-chlamydial IgG. T lymphocytesbearing the and antigen receptors were present in every semensample. Men with seminal anti-chlamydial IgA, however, had significantly(P = 0.035) elevated semen T cell concentrations (median 3100cells/ml) than did men lacking this antibody (median 1400 cells/ml);concentrations of T cells were comparable in both groups. Genitaltract sperm autoimmunity, as shown by antibodies bound to motileejaculated spermatozoa, was detected in 13 (27.1%) men. Thepresence of these antibodies was associated with elevated concentrationsof both (median 4200 versus 700 cells/ml) and (median 5000versus 850 cells/ml) T cells (P = 0.0002 and 0.0001 respectively).Men with antisperm antibodies only in their serum had seminalT cell concentrations comparable with men testing negative forantisperm antibodies. Anti-chlamydial IgA was identified insemen from four of 10 men with IgA bound to their spermatozoaand in none of the men with only spermatozoabound IgG. Therewas no relationship between sperm quality and the occurrenceof seminal IgA antibodies to either C.trachomatis or spermatozoa.An asymptomatic C.trachomatis infection activates T cells withinthe male genital tract, which may lead to antisperm antibodyformation and immune-mediated infertility.     

Andrology: Insulin-like growth factor-I and {alpha}2-macroglobulin in seminal plasma correlate with semen quality

Insulin-like growth factor-I (IGF-I) and ₂-macroglobulin (₂-M)are believed to be involved in the development of germ cells.IGF-I is mainly controlled by concentrations of human growthhormone (HGH), influences cell proliferation and differentiationand its action is mediated by insulin-like growth factor-bindingproteins (IGFBP), placental protein 14 (PP14) and prostate-specificantigen (PSA). ₂-M acts as a broad spectrum proteinase inhibitorand a binding protein for many cytokines and hormones, e.g.inhibin and activin. This study was designed to identify concentrationsof these molecules in seminal plasma in normal semen samplesof healthy men, correlations with semen quality, the relationshipof IGF-I and ₂-M with factors affecting male fertility, andwhether vasectomy influences the concentrations of these molecules.Concentrations of IGF-I and₂-M in human seminal plasma wererelated to semen quality, basal concentrations of HGH, testosterone,IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 inseminal plasma and to serum concentrations of luteinizing hormone(LH) and follicle stimulating hormone (FSH). Commercially availableassays were used to analyse 69 semen samples of various qualityand 11 post-vasectomy samples. IGF-I concentrations in seminalplasma were significantly correlated with the percentage ofmorphologically normal spermatozoa (r = 0.748, P = 0.00001)and sperm concentration (r = 0301, P = 0.011), but negativelycorrelated with serum FSH (r = –0.506, P = 0.00006) andPSA in seminal plasma (r = –0388, P = 0.0009). Total ₂-Mwas significantly correlated with sperm count (r = 0.423, P= 0.0005), percentage of progressively motile spermatozoa (r= 0.444, P = 0.00019), quality of motility (sperm motile efficiency,r = 802, P = 0.00001) and straight line velocity (r = 0.411,P = 0.0013). Correlation between the sperm concentration andHGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomyreduced the concentration of total ₂-M (P = 0.00008) and HGH(P = 0.0068) in the seminal plasma; IGF-I was also reduced aftervasectomy when the total ejaculate amount was considered. ThusIGF-I and ₂-M are significant for the germ cell development:IGF-I hi the maturation of spermatozoa and ₂-M in progressivemotility.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

A comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection

The objective of this study was to assess fertilization, implantationand pregnancy rates in infertile patients with severe teratozoospermia[P (poor prognosis) pattern sperm morphology assessed by strictcriteria] treated by in-vitro fertilization (IVF) using a highinsemination concentration (HIC), or by intracytoplasmic sperminjection (ICSI). This was a retrospective cohort study performedin an academic tertiary institution. The outcome of 115 consecutiveICSI cycles was compared to that of a similar number of cyclesof IVF with HIC performed during a similar time frame and matchedby woman's age and basal serum (cycle day 3) follicle stimulatinghormone concentrations. The inclusion criteria were sperm morphology4% normal forms (P pattern) and 1 x106 total motile spermatozoaper ejaculate. The diploid fertilization rate in the HIC-IVFgroup was 86% and in the ICSI group 68% (P < 0.05). Importantly,an equal number of embryos was transferred to both groups ofpatients. The morphological quality of the embryos (proportionof transfers having superior morphology embryo scores) was significantlybetter in the ICSI group than in the patients receiving HIC-IVF.Although there was a clear trend for better implantation andpregnancy rates in the ICSI group, these differences were notstatistically significant We conclude that, although HIC-IVFresulted in a higher fertilization rate than ICSI in patientswith severe teratozoospermia, ICSI produced a significantlyhigher proportion of morphologically superior embryos with atendency towards a higher implantation potential. Therefore,teratozoospermic patients having adequate numbers of motilespermatozoa should be offered ICSI as an alternative to modified(HIC) IVF treatment.     

Evaluation of {beta}-endorphin and interleukin-6 in seminal plasma of patients with certain andrological diseases

Human semen contains large amounts of opioid peptides and cytokines.We have measured the concentrations of interleukin (IL)-6 in140 semen samples and of -endorphinin 77 semen samples. Themedian concentration of endorphinin seminal plasma from normozoospermicmen(n = 23) was 154.7 pg/ml (10th—90th percentiles, 42.0—774.6),and there was no significant difference in the -endorphin concentrationamong normozoospermic, oligozoospermic (n= 28), asthenozoospermic(n= 15), azoospermic(n= 4) and post-vasectomy (n= 7) samples.There was no correlation between -endorphin concentration andsperm characteristics, nor with blood hormones. Endorphinconcentration was lower in cases with immunelogical infertility,as revealed by a positive direct mixedantiglobulin reactiontest (n = 12) ( > 0.01), than inmatched controls. The medianconcentration of IL-6 insamples with normal sperm concentration,motility andmorphology with or without white blood cells (n=39) was 26.1 pg/ml (10th–90th percentiles, 7.3–172.3),and there was no significant difference in the IL-6 concentrationamong normozoospermic, oligozoospermic (n= 46),asthenozoospermic(n= 32), azoospermic (n= 13) and post-vasectomy (n= 10) samples.The IL-6 concentration was significantly higher in cases ofvaricocele (n= 22)without white blood cells in semen (P <0.001) than in matched controls without varicocele (n= 23).In addition, the IL-6 concentration was elevated (P < 0.0001)in cases with accessory sex gland inflammation (n= 40). IL-6concentration was positively correlated with white blood cellsin semen (n= 60, r = 0.59, P < 0.0001), but there was nocorrelation with -endorphin concentration. The IL-6 concentrationchosen to differentiate between cases with and without accessorygland inflammation was 45.3 pg/ml, with a specificity of 80.6%and a sensitivity of 92.5%. It is concluded that -endorphinin seminal plasma playsan immune suppressive role, and thatincreased IL-6 concentration may be related to testicular dysfunctionincases with varicocele. Furthermore, IL-6 is an accurate markerof accessory sex gland inflammation.     

Reply: Comment on: Is progesterone elevation on the day of human chorionic gonadotrophin administration associated with the probability of pregnancy in in vitro fertilization? A systematic review and meta-analysis. By Venetis et al (2007)

Sir, We would like to thank Dr Bosch for his interest in our work(Venetis et al., 2007). Dr Bosch raises certain issues regardingthe interpretation of the results of this systematic reviewand meta-analysis, which deserve commenting. (i) The concept of trend In the systematic review and meta-analysis by Venetis et al.(2007), a clear research     

In-vitro fertilization in the presence of antisperm antibodies detected by the mixed antiglobulin reaction (MAR) and the tray agglutination test (TAT)

Data from 33 couples suffering from male immune infertility,who underwent 47 in-vitro fertilization (TVF) cycles betweenJanuary 1989 and August 1991, were retrospectively analysed.The serum of all the 33 male partners had elevated tray agglutinationtest (TAT) titres ( 1: 16) and positive mixed antiglobulin reaction(IgG MAR) test results in their semen. There was a slight correlationbetween these tests in semen and serum. Fertilization rateswere analysed in three sperm MAR subcategories. Only the stronglypositive MAR group (values 90%) revealed a significant reductionin fertilization rate compared to the other MAR groups. However,this was not observed with increasing serum TAT titres. Fertilizationrates were decreased in asthenozoospermic (20.1%) compared tonormozoospermic (34.0%) male partners. This occurred also withcouples not affected by immunological factors, but when antispermantibodies were present, the fertilization rates were significantlypoorer irrespective of whether the sperm motility was normalor decreased. Once fertilization occurred, the pregnancy ratewas not affected by the severity of immunological factors. Inassisted reproduction the sperm preparation techniques may reducethe inhibiting effects of antibodies bound to the spermatozoa,and when there are several oocytes to be inseminated, the chanceof fertilization rises.     

Integrins are not involved in the process of human sperm-oolemmal fusion

BACKGROUND: We investigated whether integrins are required forthe human sperm–oocyte binding and fusion processes. METHODS:The expression of several integrin subunits at the human oocyteplasma membrane was investigated using immunofluorescence microscopy,and the functional role of integrins expressed at the humanoocyte surface in sperm–oocyte interaction was studiedusing a zona-free human oocyte binding and fusion assay. A totalof 144 unfertilized oocytes were stained with anti-integrinantibodies and 147 zona-free unfertilized oocytes were inseminatedin the presence of various anti-integrin antibodies that wereexpressed in oocyte plasma membrane. RESULTS: The antibodiesof six integrin subunits (₂, ₃, ₅, ₆, _V, _M) and six integrinsubunits (₁, ₂, ₃, ₄, ₅, ₆) were bound to the surface of fixedunfertilized oocytes. In contrast, the presence of ₁ and ₄ subunitscould not be verified. The human sperm–oocyte bindingwas only partially inhibited by blocking antibodies of ₂, ₃,₅, ₆, _V, _M, ₁, ₂ and ₃ with a maximum of 55% inhibition, butantibodies of ₄, ₅ and ₆ showed no effect on sperm–oolemmalbinding. A similar reduction of the number of fused sperm wasobserved. However, the ratio of fused sperm to total sperm (boundand fused) was not impaired by all integrin antibodies, suggestingthat integrins had no role in the sperm–oolemmal fusionprocess. CONCLUSIONS: These results suggest that one of thebinding mechanisms can be inhibited by integrin antibodies butthat this mechanism does not play an essential role in the humansperm–oolemmal binding and fusion processes. The othermechanisms, insensitive to integrins, may involve both bindingand fusion processes in human oocytes.     

Andrology: Analysis of the extent to which sperm movement can predict the results of ionophore-enhanced functional assays of the acrosome reaction and sperm-oocyte fusion

This study has examined the extent to which the informationgenerated by ionophore-enhanced bioassays of the acrosome reactionand sperm-oocyte fusion might be predicted from the computer-aidedanalysis of sperm motility Strong correlations (r 0.7) wereobserved between specific components of sperm movement in semenand the potential for A23187-induced sperm-oocyte fusion, generatinga stepwise regression coefficient of R = 0.663 on the basisof two criteria, percentage progressive motility and amplitudeof sperm lateral head displacement (ALH). The movement characteristicsof the spermatozoa recovered from the Percoll gradients gavean even higher R value of 0.838 on the basis of four variables(percentage rapid, average path velocity, straightness and ALH).In contrast, the ability of human spermatozoa to undergo acrosomereaction in response to A23187 exhibited a limited correlationwith sperm movement, whether these measurements were made inthe original semen sample or following Percoll purification(R 0.4). These results have diagnostic implications, sincesperm-oocyte fusion and the acrosome reaction clearly differin their relative dependence on sperm motility. In practicalterms, it should be noted that the computer-aided analysis ofsperm movement was shown to provide up to 70% of the informationgenerated by the more laboured assessment of sperm-oocyte fusion.     

Treatment of spermagglutination with proteolytic enzymes. I. Sperm motility, vitality, longevity and successful disagglutination

The treatment of antibody-mediated spermagglutination by corticosteroidtherapy has a high incidence of side-effects and sperm washingis often followed by re-agglutination. The possibility of enzymaticdisagglutination was therefore investigated. In the first partof the study the effects of four proteases on sperm motility,vitality and longevity were evaluated. Subtilisin had prohibitivelydetrimental effects even at 10 U/ml. However, chymotrypsin (500U/ml), trypsin (500 U/ml) and papain (50 U/ml) had no adverseeffects. In the second series of experiments one or more ofthese latter three enzymes was found to disagglutinate spermatozoawhich had previously been incubated with sperm-agglutinatingantibody-positive sera in 87% of cases. Although further investigationis required, enzymatic disagglutination may be beneficial forthe treatment of immunologically mediated spermagglutination.     

Acrosin activity in human spermatozoa in relation to semen quality and in-vitro fertilization

Acrosin, a sperm proteinase released during acrosomal exocytosis,facilitates penetration through the oocyte vestments. The purposeof this investigation was to determine if a correlation existsbetween the acrosin activity of ejaculated human spermatozoa,before motility enrichment techniques, and in-vitro fertilization(IVF) success using selected (glass wool or swim-up) spermatozoa.Since all the oocytes were retrieved from women receiving exogenoushormonal stimulation and a mixed population of mature and immatureoocytes were encountered, only cases with 50% mature oocyteswere analysed. Under these conditions, the acrosin activitywas significantly greater (P < 0.01) in the ejaculates inwhich spermatozoa ultimately fertilized <70% of the matureoocytes, than in the ejaculates in which spermatozoa ultimatelyfertilized 70% of the mature oocytes. Furthermore, a strongcorrelation (r = 0.962, P = 0.0001) was detected between pre-IVFacrosin activity and subsequent high (70%) IVF success. Acrosinactivity from normozoo-spermic and oligoasthenozoospermic menwas also compared and was significantly (P < 0.01) higherfor the normozoo-spermic group. These data suggest that measurementof acrosin activity may be a valuable clinical laboratory assayfor assessing the sperm fertilizing potential and that low acrosinactivity is associated with abnormal semen characteristics.     

Female age predicts embryonic implantation after ICSI: a case-controlled study

From 1 October 1991 until 31 December 1993, 1270 cycles forintracytoplasmic sperm injection were performed. Of these, 71(5.6%) were carried out in women 40 years of age. The semencharacteristics in couples40 years of age or <40 years weresimilar. The mean male age for the older group of women was47.1 years (range 34–67) versus 35 years (range 25–71)for the younger group of women (P <0.001). The mean femaleage was 41.9 years (range 40–-47) and 31.8 years (range23–39). The numbers of cumulus—oocyte complexesand metaphase-II oocytes were significantly lower in women 40years of age (P < 0.001). The mean numbers of replaced embryoswere respectively 2.3 (133/59) in women 40 years of age and2.5 (160/63) in women <40 years of age. The delivery rateper retrieval and per transfer was significantly lower in women40 years of age (P < 0.05). The delivery rates per retrievaland per transfer were respectively 7% (5/71) and 8.5% (5/59)in the older group of women versus22.5% (16/71) and 25.4% (16/63)in the younger group. Female age is the predictive factor forembryonic implanatation.     

Andrology: The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa

The usefulness of sub-zonal injection (SZI) for the treatmentof severe male factor infertility has been restricted by lowand unpredictable fertilization rates and the high risk of polyspermyafter the injection of multiple spermatozoa. In this prospectivestudy, we have evaluated whether sperm morphology and the percentageof acrosome-reacted spermatozoa at the time of injection canbe used to predict SZI fertilization outcomes. Populations ofmotile spermatozoa equivalent to those injected were collectedfrom the medium/oil interface immediately after SZI of eachcohort of oocytes. Morphology was assessed using the World HealthOrganization 1987 criteria and the acrosomal status of spermatozoawas determined after staining with rhodamine-conjugated Pisumsativum agglutinin. A fertilization index (FI) was calculatedto express the actual fertilizing potential of the spermatozoainjected. In all, 67 patients underwent 72 SZI cycles. The overallfertilization and polyspermy rates were 36 and 47% respectively,and a clinical pregnancy rate per transfer of 22% was achieved.Linear regression analysis demonstrated a statistically significantrelationship between morphology and the FI (r = 0.506, P <0.0001). Patients with <10% normal morphology always hada FI < 10%, and this was reflected by low fertilization andpolyspermy rates and the high number (32%) of cycles with completefailure of fertilization in this group. In patients with >10% normal morphology, there were two patterns: low (10% FI)or high (>10% FI) fertility. This was evident in the fertilization(23 and 85%, respectively) and polyspermy (25 and 68%, respectively)rates of these two patient sub-groups. While the percentageof acrosome-reacted spermatozoa at the time of injection wasweakly correlated with the FI (r = 0.292, P < 0.05), it couldnot be used to predict differences in fertilization potentialbetween patient sub-groups. We conclude that sperm morphologyand acrosomal status at the time of injection are of limiteduse in predicting SZI fertilization outcomes, although patientswith poor morphology ( 10% normal) have lower fertilizationand polyspermy rates.     

Genetics: Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and {Delta}F508 mutations

W1282X (W) and F508 () are the two most common mutations ofthe cystic fibrosis Israeli population. Patients who are homozygotes(WW and ) as well as compound heterozygotes (W) present a severephenotype of the disease. In the present study, we have developeda polymerase chain reaction (PCR)-based method for the detectionof both mutations simultaneously in a single blastomere. Unfertilizedhuman oocytes and single polyspermic blastomeres were subjectedto a two-round PCR amplification: a first round of multiplexPCR followed by a second round of nested PCR, done seperatelyat each locus. Clear signals at both loci were obtained in 51%(47/65) of oocytes and 69% (24/35) of blastomeres. The genotypeof the single cell analysed was determined by endonuclease digestionof the W products and by heteroduplex formation of the F products.This diagnostic system will allow the identification of affectedembryos (WW, , W) as well as phenotypically normal carriers(W++), and therefore may be used for cystic fibrosis preimplantationdiagnosis in families who carry either or both mutations