Severe Asthenozoospermia Research Articles

Two cases of sperm immotility: a mosaic of flagellar alterations related to dysplasia of the fibrous sheath and abnormalities of head-neck attachment

To characterize the association of two systematic sperm defects. Case report. University, Interdepartmental Centre for Research and Therapy of Male Infertility. Patient 1, 42 years old, and patient 2, 38 years old, both with severe asthenozoospermia. Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, immunocytochemistry for tubulin, and fluorescence in situ hybridization (FISH) for chromosomes 18, X, and Y. Admixture of dysplasia of the fibrous sheath (DFS) and head-tail misalignment up to acephalic sperm detected by microscopic methods. In both patients, DFS was present in incomplete form and was associated with acephalic sperm and abnormal head-tail attachment. In patient 2, spermatozoa were also affected by necrosis that may cause fragmentation leading to short flagella; submicroscopic examination allowed defining only the origin of these "stumpy" tails. Immunofluorescence confirmed the sperm alterations. FISH revealed an altered frequency of diploidy and disomy in patient 2 and a slight increase in diploidy in patient 1. The importance of ultrastructural sperm evaluation for correct identification of sperm pathologies is evident, particularly regarding assisted reproduction technology and genetic risk assessment.

Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia

To explore the contribution of an altered structure of sperm mitochondria to human asthenozoospermia. A retrospective study. Andrology Clinic, University of L'Aquila. Fifteen ejaculates with forward motility (FM) ≥ 50%, and 57 asthenozoospermic ejaculates (FM <50%, sperm vitality >50%), including 14 ejaculates with systematic genetic defects of tail principal piece, and 43 ejaculates with unexplained asthenozoospermia. Fifty sections of tail middle piece (MP) were blindly analyzed by transmission electron microscopy in each ejaculate for normal mitochondrial membrane organization, after exclusion of tails with disrupted cell membranes. Percentage of MPs with normal mitochondrial membranes (% normal MPs). Percent normal MPs showed a strong correlation with forward motility. Variation of % normal MPs explained a 45% variation of sperm motility at multivariate linear regression analysis, confirming the strong association between the two parameters in a population including ejaculates with normal motility and with unexplained asthenozoospermia. Percent normal MPs was significantly reduced in severe unexplained asthenozoospermia (FM <10%; n = 16) compared with samples with normal motility (FM ≥ 50%; n = 15); 21% (10.5%-38%) and 68% (52%-73%), respectively. Structural defects in mitochondrial membranes represent a main feature of severe unexplained asthenozoospermia.

A sperm viability test using SYBR-14/propidium iodide flow cytometry as a tool for rapid screening of primary ciliary dyskinesia patients and for choosing sperm sources for intracytoplasmic sperm injection

Spermatozoa viability tests based on dual-color flow cytometry after staining with Sybr-14/propidium iodide were performed on 44 men with complete asthenospermia for primary ciliary dyskinesia (PCD) screening, and seven were identified with PCD by electron microscopy of ultrastructural ciliary defects. Six PCD patients underwent eight intracytoplasmic sperm injection therapy cycles using ejaculated sperm or testicular sperm, obtaining a mean fertilization rate of 46.6%, with three healthy babies born and one in utero at the time of writing.

A case of severe asthenozoospermia: a novel sperm tail defect of possible genetic origin identified by electron microscopy and immunocytochemistry

To characterize a novel flagellar defect involving 98% of sperm tails. Case report. Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. A 45-year-old infertile man with severe asthenozoospermia. Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization (FISH) for chromosomes 9, 16, 18, X, and Y. Flagellar abnormalities detected by microscopical methods. An apparent heterogeneity was observed: extremely elongated tails prone to ruptures; coiled tails at different levels with a strongly rolled axoneme or with a curl in the final flagellar segment; and V-shaped, isolated, bent tails. Transmission electron microscopy revealed the presence of normal heads, disorganized flagellar structures, and dynein deficiency. The FISH analysis was normal. We report a new sperm defect, characterized by abnormal elongation of the tail, which was prone to ruptures at different levels, concomitant with coiled tails, which were impossible to measure in length. This defect remained constant in different examined ejaculates and applied to the entire sperm population of a sterile man, the son of first-degree cousins, indicating a potential genetic origin.

The oocyte activation and Ca2+oscillation-inducing abilities of mouse and human dead (sonicated) spermatozoa

In ICSI procedures, it is well known that the selection of viable (live) spermatozoa and certain types of immobilization prior to injection is very important for obtaining successful results, but unfortunately there are rare situations when only immotile spermatozoa are available (such as in severe asthenozoospermia or necrozoospermia). In such cases, failure of oocyte activation after ICSI often occurs and may be due to the lack of SOAF (sperm-borne oocyte activating factor) activity. In order to investigate the SOAF activities of dead spermatozoa, mouse and human spermatozoa were immobilized (killed by sonication), maintained in THF medium for varying time intervals (up to 72 h) and then injected into mature unfertilized mouse oocytes. Injected mouse oocytes were examined for their activation, development into blastocysts and Ca2+ responses by imaging and confocal laser scanning microscope. The rates of oocyte activation, blastocyst development and normal patterns of Ca2+ oscillation from the killed-sperm-injected oocytes decreased gradually in accordance with the maintenance interval between sonication and injection. For injection with mouse sonicated spermatozoa, the rate of normal Ca2+ oscillations declined first (after a 3 h maintenance interval) and then blastocyst development was gradually obstructed (after approx. 10 h). The oocyte activation-inducing ability of dead spermatozoa was maintained for a relatively long period, but began to decline after 20 h. The activation rates and Ca2+ response of the oocytes that were injected with human sonicated spermatozoa decreased earlier than those injected with mouse spermatozoa. Although the oocyte activation-inducing ability was maintained for a relatively long time after the death of the spermatozoa, embryo development into blastocysts and the rate of normal Ca2+ oscillations declined after a short maintenance interval between sonication and injection. The Ca2+ response seemed to be the most sensitive indicator for the evaluating the SOAF activity of dead (killed) spermatozoa.

Abnormal elongation of midpiece, absence of axoneme and outer dense fibers at principal piece level, supernumerary microtubules: a sperm defect of possible genetic origin?

To characterize a flagellar defect involving 95% of the sperm population from an infertile man. Case report. Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. A 42-year-old infertile man with severe asthenozoospermia. Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM), tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization for chromosomes 18, X, and Y. Ultrastructural abnormalities of the flagellum detected by methods listed. Ultrastructural analysis revealed, in 95% of sperm cells, the total absence of the axoneme and outer dense fibers at the principal piece level, whereas the midpiece appeared abnormally long. Tubulin localization showed a total disorganization of the axoneme with a network of microtubular structures emerging randomly at any level of the flagellum. Fluorescence in situ hybridization analysis was normal. We report a rare sperm tail defect, characterized by abnormal elongation of the midpiece and absence of the axoneme and the outer dense fibers at the principal piece level in 95% of flagella. This defect occurs in the vast majority of the sperm population from a sterile man, and therefore a genetic origin could be hypothesized.

Results of intracytoplasmic sperm injection in two infertile patients with abnormal organization of sperm mitochondrial sheaths and severe asthenoteratozoospermia

To report assisted reproduction technologies (ART) outcome and characterize severe mitochondrial sheath (MS) anomalies in two infertile asthenoteratozoospermic patients. Case reports. Private IVF clinic and academic research institution. Two infertile men with asthenoteratozoospermia. Intracytoplasmic sperm injection (ICSI) was performed in both cases. Clinical and laboratory evaluation were performed and spermatozoa studied by epifluorescence microscopy and transmission electron microscopy (TEM). Patient 1 had sperm with acute bendings at the level of the narrow midpieces. Mitochondria were either scarce or absent. Three ICSI embryos were transferred. A pregnancy was achieved followed by a miscarriage at the end of the first trimester. Patient 2 had sperm with very long MSs. The number of gyres was increased to more than 30. Two ICSI cycles were performed with good fertilization rates and embryo quality, but no pregnancy was achieved. MS defects were studied by phase-contrast, epifluorescence microscopy, and TEM that afforded a detailed view of the sperm midpiece and the topography of the whole flagellum. The results indicate that midpiece defects, while causing severe asthenozoospermia and lower fertilizing potential, may not necessarily represent negative prognostic factors in ART.

Positive expression of the immunoglobulin superfamily protein IZUMO on human sperm of severely infertile male patients

Recently, the immunoglobulin superfamily protein IZUMO on the sperm surface was identified as being essential for sperm-egg fusion. Although we examined the expression of IZUMO on sperm of 13 male infertile patients with severe oligozoospermia and/or athenozoospermia and 12 infertile patients with fertilization failure in previous conventional IVF to determine whether it is varied in infertility patients, we could not identify any patients with IZUMO negative by immunocytochemistry.

Utrastructural analysis of asthenozoospermic ejaculates in the era of assisted procreation

To define by transmission electron microscopy (TEM) analysis, the prevalence of sperm tail defects of genetic origin among men suffering for a reduced or absent sperm motility. A retrospective study. Andrology Clinic, University of L'Aquila, Italy. The 120 ejaculates of infertile men with a forward motility (FM) < or =20% were compared to 200 ejaculates with a FM >20%. All ejaculates had a sperm vitality >50%. Some 25-50 tails were analyzed by TEM in each ejaculate. Receiver operating characteristics (ROC) analysis was applied by plotting the FM of cases with or without genetic tail defects detected by TEM. The area under the ROC curve was 0.96 (95% confidence interval 0.92-0.98). The positive predictive accuracy for genetic tail defects in ejaculates with 0% FM was 46%. Three of 17 cases with genetic tail defects were classified as false negative when TEM analysis was restricted to ejaculates with 0% FM. A FM < or =7%, allowed the identification of all cases with genetic tail defects. The TEM analysis identifies sperm tail defects of genetic origin and should be restricted to ejaculates with severe asthenozoospermia (< or =7% motile sperm) and sperm vitality >50%.

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: asthenozoospermia

The aim of aneuploidy evaluation in spermatozoa from patients presenting spermatogenesis defects is to identify a relationship between meiotic errors and quantitative or qualitative alterations of spermatogenesis. During the past ten years, the use of fluorescence in situ hybridization (FISH) has permitted the determination of the frequency of numerical chromosome aberrations in different clinical situations. It has been established that infertile males with reduced sperm count and a normal constitutional karyotype have a significantly high risk of aneuploidy in their spermatozoa particularly regarding sex chromosomes. Concerning sperm motility, the data are more controversial. However, patients of severe asthenozoospermia induced by specific morphological deformities involving sperm flagella have a significantly high risk of producing aneuploid spermatozoa.

An ultrastructural and immunocytochemical study of a rare genetic sperm tail defect that causes infertility in humans

Objective To characterize and describe the ontogenesis of a rare flagellar defect affecting the whole sperm population of a sterile man. Design Case report. Setting Regional referral center for male infertility in Siena, Italy. Patient(s) A 28-year-old man with severe asthenozoospermia. Intervention(s) Physical and hormonal assays, semen analysis, and testicular biopsy. Main outcome measure(s) Semen samples and testicular biopsies were analyzed by light and transmission electron microscopy; immunocytochemical study with anti-β-tubulin and anti-AKAP 82 antibodies was performed to detect the presence and distribution of proteins. Result(s) Ultrastructural analysis of ejaculated spermatozoa and testicular biopsy revealed absence of the fibrous sheath in the principal-piece region of the tail. Fibrous sheath-like structures were observed in cytoplasmic residues and residual bodies released by spermatids in the seminiferous epithelium. Other anomalies observed were supplementary axonemes and mitochondrial helix elongation. These features were confirmed by immunocytochemical staining. Conclusion(s) This rare sperm tail defect, characterized by absence of the fibrous sheath, presence of supplementary axonemes, and an abnormally elongated midpiece, originates in the seminiferous tubules during spermiogenesis, as detected in testicular biopsy sections. These defects occur in the whole sperm population, and therefore a genetic origin could be suggested.

Diversity of the inhibitory effects on fertilization by anti-sperm antibodies bound to the surface of ejaculated human sperm.

The presence of anti-sperm antibodies (ASA) in males can reduce fecundity. However, it has been shown that there is a diversity of ASA bound to the sperm surface. This study was performed to investigate the inhibitory effects on fertilization by ASA in males. ASA were detected using the direct-immunobead test (D-IBT) in 509 semen samples. In some cases, the direct-sperm immobilization test (D-SIT) was carried out. The fertilizing ability of infertile males with ASA was determined as follows; (i) an IVF fertilization rate of >/=50%, (ii) a hemizona index (HZI) of >/=50%, and (iii) pregnancy established without the use of ART. In total, 18 (3.54%) infertile males had ASA on the sperm surface. Except for one male with an absolute indication for ICSI because of severe asthenozoospermia and two males who dropped out of this study, fertilizing ability in 15 males could be determined. Four (26.7%) men did not satisfy the criteria. The existence of sperm immobilizing antibodies on the surface of ejaculated sperm had no impact on fertilization. In four (57.1%) of seven patients who had IB-bound sperm of >/=80%, fertilizing ability was inhibited, while none of the eight patients who had <80% IB-bound sperm had an inhibitory effect on fertilization. There was a significant difference between the two groups (P = 0.01). Some sperm-bound antibodies are related to the inhibitory effects on fertilization, indicating that a diversity of sperm-bound antibodies exists in males. This result might be one of the reasons for the controversy of the relationship between ASA and male immunological infertility. Based on the present study, a sperm-zona pellucida binding assay should be performed for appropriate decision making in infertile males with ASA.

The effect of pentoxifylline (PF) in severe asthenozoospermia treatment on the results of ICSI program

Objective: Intracytoplasmic sperm injection (ICSI) is now widely used for male factor treatment in human IVF/ET program. Although poor sperm parameters including count, sperm motility and morphology has been reported not important in ICSI program, sperm motility is a prerequisite indicator to find viable spermatozoa in the treatment of severe asthenozoospermia. Pentoxifylline (PF) and other methylxanthines are known to influence sperm metabolism and motility. Because of its enhancing effect on sperm motility and its role in facilitating the acrosome reaction, the agent is now commonly used in human ICSI program to easily find motile spermatozoa. It has been postulated that PF, being a phosphodiesterase inhibitor, increase sperm kinetic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. The aim of study was to evaluate the effect of PF on the conventional ICSI program undergone in severe asthenozoospermia.

Successful pregnancy with intrauterine insemination using vasal sperm retrieved by electric stimulation

Objective: To determine the feasibility of electric stimulation applied to the epididymis or vas deferens to retrieve vasal sperm. Design: Two case reports. Setting: Assisted reproduction practice in a hospital. Patient(s): Two patients with retrograde ejaculation and severe asthenozoospermia. Intervention(s): Pulse electric simulation was applied to the epididymis or vas deferens to retrieve sperm via a tube canulated into the vas deferens. After diluting with culture medium, retrieved sperm were used for IUI. Main Outcome Measure(s): The volume of retrieved vasal fluid and motility parameters of the sperm, the achievement of pregnancy, and outcome of pregnancy. Result(s): In patient 1, the volumes of vasal fluids and numbers of sperm were 10–40 μL and 31.4–75.9 × 10 6, respectively, during two cycles. Sperm motility was 88.4%–93.2%. His wife became pregnant and was delivered of a healthy infant by IUI during the second cycle. In patient 2, 0.6 × 10 6 sperm with a motility of 70% were retrieved and used for IUI. However, his wife did not become pregnant. Conclusion(s): Electric vasal sperm retrieval is a feasible method for collecting sperm from the vas deferens in selected patients with male factor infertility.

Successful ICSI in a case of severe asthenozoospermia due to 93% non-specific axonemal alterations and 90% abnormal or absent mitochondrial sheaths

The use of IVF and intracytoplasmic sperm injection (ICSI) to treat male infertility due to severe asthenozoospermia in cases of electron microscopically confirmed axonemal abnormalities has been reported. However, even with these sophisticated technologies, very few pregnancies and births have been achieved with ejaculated spermatozoa in infertility due to this condition. The existence of sperm-derived defects preclude normal embryo development, and another reason for low success rates may be that the most commonly used method for selecting viable spermatozoa for ICSI is sperm motility, which is absent in affected men. Consequently, the likelihood of selecting non-viable spermatozoa for ICSI is higher in these cases. This report describes a case of a pregnancy achieved by ICSI with ejaculated spermatozoa containing 93% non-specific axonemal alterations and 90% abnormal or absent mitochondrial sheaths. A total of 14 oocytes was obtained from the 31-year-old patient in her first IVF treatment cycle. Three of the 13 matured (metaphase II) oocytes were inseminated conventionally, and the other 10 metaphase II oocytes were submitted to ICSI. None of the oocytes inseminated conventionally were fertilized. Of the 10 oocytes submitted to ICSI, four zygotes and cleavage embryos resulted. Three embryos were transferred on day 3, and a successful pregnancy was achieved. One gestational sac, together with regular heart activity, was recorded by ultrasonography at 8 weeks of gestation. A pregnancy is currently ongoing.

The effects of follicular fluid and platelet-activating factor on motion characteristics of poor-quality cryopreserved human sperm.

To evaluate the effects of follicular fluid and platelet-activating factor on sperm motion characteristics of cryopreserved oligospermic and normospermic samples. Sperm motion characteristics were evaluated prior to cryopreservation, immediately after thawing and following incubation in human tubal fluid, follicular fluid, or 1-microM platelet-activating factor cultures. Sixteen oligospermic samples and 20 normospermic samples were examined. Sperm motion characteristics were analyzed manually according to WHO criteria (1999) and also with an automated videomicrography system. Incubation in follicular fluid increased overall motility and the percentage of sperm with fast progressive motility in normospermic but not oligospermic samples. Incubation with platelet-activating factor increased overall motility and the percentage of sperm showing nonprogressive motility in both oligospermic and normospermic samples. The stimulatory effects of culture in follicular fluid as seen in normospermic samples do not show a significant benefit in oligospermic cryopreserved samples. Platelet-activating factor and follicular fluid increase motility via different mechanisms. Incubation of oligospermic cryopreserved sperm with PAF increases the number of motile sperm, thereby enabling easier identification of viable sperm for intracytoplasmic sperm injection in samples with severe asthenozoospermia.

Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men.

An ultrastructural study of spermatozoa in a series of 247 severely asthenozoospermic patients disclosed two kinds of anomalies. The first was dysplasia of the fibrous sheath, a primary defect of spermatozoa with hypertrophy and hyperplasia of the fibrous sheath, associated axonemal anomalies, familial incidence and chronic respiratory disease. The patients could be divided into two subgroups: the complete form (all spermatozoa affected) and the incomplete form (alterations in 70-80% spermatozoa). There were no spontaneous or in-vitro fertilization (IVF) pregnancies. Intracytoplasmic sperm injection (ICSI) in six patients resulted in successful fertilizations, but only two pregnancies were obtained. These features configure a phenotype that suggests a genetic origin. The second anomaly was non-specific flagellar anomaly (NSFA), random secondary flagellar alterations affecting variable numbers of spermatozoa, without respiratory disease or familial incidence. 54 men with NSFA were followed for 2-6 years. Of these, 18 achieved conception, either spontaneous or by means of assisted fertilization, followed by 14 pregnancies and 12 live births. Their sperm motility significantly increased during the follow-up period. In the remaining 36 men motility did not change during the follow-up period and there were no fertilizations or pregnancies. We conclude that in severe asthenozoospermia, ultrastructural examination of spermatozoa has an effective prognostic value, identifying two syndromes with very different flagellar alterations and fertility potentials.

Spontaneous Pregnancy in Couples Waiting for Artificial Insemination Donor Because of Severe Male Infertility

Abstract Objective: To assess the spontaneous fertility in couples with severe seminal conditions while waiting for artificial insemination donor. Study design: Prospective follow-up during a period of 24 months. Setting: University Medical School. Participants: There were 285 couples in which the male had a very severe seminal pathology: 166 azoospermia, 86 oligozoospermia and 33 severe asthenozoospermia. Outcome measures: Pregnancy rates after being included on waiting list. Results: The spontaneous pregnancy rate was 3.2% ( 9 285 ), per month spontaneous pregnancy rate being 0.13%. Spontaneous pregnancy rate was 0% in azoospermia ( 0 166 ), versus 7.6% ( 9 119 ) in non-azoospermia cases. Spontaneous pregnancy rate was 8.5% ( 4 47 ) in the group with less than 0.1 million motile sperm/cc, 6.5% ( 3 46 ) in the group between 0.1 and 1 million/cc and 7.7% ( 2 26 ) in the group with 1–2 million/cc. Conclusion: In a 2-year follow-up, pregnancy rate among non-azoospermic couples before undergoing artificial insemination was 7.6%. Extramatrimonial pregnancy (based on anamnesis and sperm analysis) seemed to be uncommon. Even in cases with less than 0.1 million of motile sperm/cc there was not a negligible spontaneous pregnancy rate

Spontaneous pregnancy in couples waiting for artificial insemination donor because of severe male infertility

Objective: To assess the spontaneous fertility in couples with severe seminal conditions while waiting for artificial insemination donor. Study design: Prospective follow-up during a period of 24 months. Setting: University Medical School. Participants: There were 285 couples in which the male had a very severe seminal pathology: 166 azoospermia, 86 oligozoospermia and 33 severe asthenozoospermia. Outcome measures: Pregnancy rates after being included on waiting list. Results: The spontaneous pregnancy rate was 3.2% ( 9 285 ), per month spontaneous pregnancy rate being 0.13%. Spontaneous pregnancy rate was 0% in azoospermia ( 0 166 ), versus 7.6% ( 9 119 ) in non-azoospermia cases. Spontaneous pregnancy rate was 8.5% ( 4 47 ) in the group with less than 0.1 million motile sperm/cc, 6.5% ( 3 46 ) in the group between 0.1 and 1 million/cc and 7.7% ( 2 26 ) in the group with 1–2 million/cc. Conćlusion: In a 2-year follow-up, pregnancy rate among non-azoospermic couples before undergoing artificial insemination was 7.6%. Extramatrimonial pregnancy (based on anamnesis and sperm analysis) seemed to be uncommon. Even in cases with less than 0.1 million of motile sperm/cc there was not a negligible spontaneous pregnancy rate

Severe asthenozoospermia: a structural and functional study

Ejaculates from 25 patients with severe asthenozoospermia (all spermatozoa immotile or only non-progressively motile) were studied to identify individually the cause of impaired motility. Multiple tests were performed, viz. light and electron microscopic studies and sperm function tests. An objective scoring was applied to both the routine and the functional analyses. Three categories of samples were identified: (1) necrozoospermia (n = 9), where sperm viability was very poor; (2) structural tail abnormality as seen by light microscopy (n = 4); and (3) ultrastructural abnormality (n = 12). In the last category, one case showed absence of dynein arms; this was associated with mitochondrial abnormalities. Mitochondrial abnormality with normal tail components was observed in the majority (n = 7) and accessory fibre abnormality in four cases. The scoring system revealed that, functionally, all samples were abnormal whereas routine analysis showed 15 samples to be subnormal and 10 to be abnormal, which indicates the need for functional analysis. Because of the multiple defects seen in these samples, there is a need for a battery of sperm function tests. This study indicates that mitochondrial defects are one of the causes that may account for the loss of sperm motility in the patient population.