Oligospermic Samples Research Articles

P-017 DNA fragmentation Index before and after density gradient centrifugation using flow cytometry, its reflection in sperm ultrastructure and its association with clinical outcome

Abstract Study question Is DNA fragmentation Index (DFI) affected by Density Gradient Centrifugation (DGC), reflected in sperm ultrastructure and associated with clinical outcome? Summary answer DFI was not affected by DGC, nor associated with clinical outcome, but it was negatively correlated with sperm concentration/motility and positively correlated with ultrastructural anomalies. What is known already Oxidative stress induces DNA fragmentation during sperm transit through the male genital tract and it is suggested to be the main mechanism causing DNA breaks after ejaculation during in vitro manipulation and sperm selection for injection/insemination. High levels of sperm DNA fragmentation have been linked to lower fertilization rates, poor embryo quality and delayed cleavage. Limited studies, have investigated in detail the effects of DGC on DFI and the reflection in sperm ultrastructure, while the predictive value of DFI on clinical outcome still remains controversial. Study design, size, duration Semen samples were collected on the day of the female partener’s oocyte collection and analysed before and after DGC for standard characteristics by light microscopy, according to World Health Organization (WHO) criteria (n = 120), for DNA Fragmentation by Flow Cytometry and for ultrastructure by Transmission Electron Microscopy (TEM). The predictive value of DFI for clinical outcome was also assessed. Participants/materials, setting, methods Standard semen analysis and DGC using the Sil-Select 40%/ 80 % layers were performed at an IVF Laboratory of an academic Hospital. Sperm DFI before and after DGC was assessed by Flow Cytometry at the Department of Immunology and Histocompatibility of the same academic Hospital. Ultrastructure analysis by TEM was carried out at an academic Histology/Embryology Laboratory following fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance DGC did not alter DFI significantly (p = 0.052), but lead to a statistically significant increase in Progressive Motility (p < 0.001). DFI was higher in men with a male factor infertility compared to men without a male factor (p = 0.032) and Oligoasthenoteratospermia (OAT) exhibited the highest DFI levels (36.38% ± 5.36). DFI was negatively correlated with sperm concentration (rho = -0.361, p < 0.01) and sperm motility (rho = -0.301, p < 0.05). No significant association was observed between DFI and clinical outcome, but statistically significant differences were observed both in the age of men (37.54±1.33 vs 42.8±1.27 p = 0.017) and the age of women (35.77±1.25 vs 40.17±0.78 p = 0.007) in the group of patients who achieved a pregmamcy and those that did not. TEM showed that DGC did not lead to an increase in ultrastructural anomalies of spermatozoa, but DFI was positively correlated with ultrastructural anomalies. In teratozoospermic men TEM revealed head, neck and tail defects, including elongated forms, axonemal defects and premature initiation of the acrosome reaction. Limitations, reasons for caution The ultrastructure analysis was not possible to be performed in severely oligospermic samples. Wider implications of the findings This study shows that sperm ultrastructure and DFI are not adversely affected by DGC confirming the safety of the preparation method for sperm selection prior to insemination/injection. Larger-scale studies are needed, to elucidate any potential correlations between DFI and the probability of pregnancy. Trial registration number not applicable

P–069 microfluidic sperm sorting vs density gradient to yield sperm with reduced DFI for patients undergoing IVF-ICSI

Abstract Study question To compare the effect of sperm preparation methods on the DFI of semen sample for patients undergoing ICSI. Summary answer On comparing the results, microfluidic sperm sorting yielded sperms with significantly less DFI as compared to density gradient method of sperm preparation. What is known already The DNA integrity of the sperm plays an important role to ensure formation of good quality embryos with increased potential of fertilization, growth and ultimately implantation.. Centrifugation has shown to add stress to the sperm and leading to DNA damage, therefore there is a need to develop techniques of sperm preparation which help in retrieving as many sperms with intact DNA from the unprocessed sample as possible. Microfludic is fluid dynamic based technique of sperm preparation. in this study, we evaluated if microfluidic sperm sorter can recover motile sperm with better DNA integrity compared to density gradient preparation method. Study design, size, duration Prospective randomized study conducted in 80 patients undergoing IVF-ICSI with normal semen parameters (based WHO criteria 2010). DFI was done using Sperm Chromatin Dispersion (SCD) test in split semen samples prepared by microfluidic sperm sorter and density gradient method. Sperm morphology and motility were also recorded and evaluated based on the WHO 2010 criteria. Participants/materials, setting, methods Semen parameters of the sample were assessed by microscopic examination. DFI of each unprocessed sample was carried out using SCD test, following that the sample was split and sperm preparation was done using microfluidic sperm sorter and density gradient. the recovered sperm were tested for DFI and the results were compared. Main results and the role of chance Mean DFI in unprocessed semen samples was 23%. the analysis of split semen samples post preparation showed that the DFI was significantly reduced with the use of microfluidic sperm sorter (mean DFI 0.6%) as compared to density gradient (mean DFI 9%). Limitations, reasons for caution A major limitation of the microfluidic sperm sorter is the use sperm concentration and motility of the semen sample. In oligospermic and asthenospermic samples, density gradient is the preferred method of preparation. Lack of data showing improvement in clinical outcomes with reduced DFI is also a major limitation. Wider implications of the findings: Microfluidics has shown to significantly reduce the DFI of the semen sample, it requires no extra equipment and cost and is relatively easy to pick up. Density gradient method of sperm preparation continues to be the preferred method due to its versatility and recovery of good quality sperm. Trial registration number Not applicable

In Silico Analysis of Sperm From Ejaculates with Different Semen Characteristics.

Background:Male infertility is associated with altered characteristics of the sperm within the ejaculate. It is possible to find molecular explanations for the observed phenotypes and their consequences. This study aimed to analyze, using a specialized software, a gene set of transcriptomic data from different types of ejaculates.Methods:Data from ejaculate samples categorized as normal, oligospermia, and teratozoospermia were obtained from Gene Expression Omnibus (GEO). After normalization, the data average for each sample category was calculated and analyzed independently using Ingenuity Pathway Analysis (IPA).Results:Five important canonical pathways are involved in normal and altered semen samples (Oligospermia and teratozoospermia) except sirtuin signaling and mitochondrial dysfunction pathways. The five most important biological processes are identified in all semen phenotypes, but the only difference is the genes connected with initiation of RNA transcription in oligospermic and asthenospermic samples.Conclusion:Surprisingly, different types of ejaculates share many pathways and biological processes; sperm proteomics as a new global approach gives clues for the development of strategies to explain the reason for observed phenotypes of ejaculated spermatozoa, their possible effect on fertility, and for implementing research strategies in the context of infertility diagnosis and treatment.

Evaluation of Semen Samples Before and After ‘Swim Up’ Technique with Mitotracker

Objectives: Infertility problems are seen in nearly 15% of married couples, and almost 50% of these involve male infertility. The sperm mitochondrion plays an important role in sperm motility. The morphology of sperm, sperm preparation techniques, and the number and motility of sperm during insemination are important parameters that affect the rate of pregnancy with intrauterine insemination. The aim of the study was to observe the changes in sperm mitochondrial activities and numbers with the Mito Tracker Red 580 kit at 30th, 45th and 60th minutes after swim up.
 Materials and Methods: In this study, we evaluated 20 oligospermic and 20 normospermic samples. The “swim-up” technique was applied to these oligospermic and normospermic samples, and changes in sperm mitochondria and number at 30, 45, and 60 min were observed using MitoTracker Red 580 staining.
 Results:The numbers of swimming sperm in oligospermic and normospermic samples were higher at 30 min than at 45 and 60 min. No marked change was detected in the mitochondrial activities of swimming sperm in oligospermic and normospermic samples at 30, 45, and 60 min after the swim-up. Our findings demonstrate that the number of swimming sperm decreased with time, but no change was seen in the mitochondrial activity of the sperm by the swim-up technique.
 Conclusion: According to findings, mitochondrial movement is not exactly related to the 30th, 45th and 60th minutes. Therefore, it is thought that it needs to be investigated in mechanisms other than mitochondrial activity.
 Bangladesh Journal of Medical Science Vol.18(3) 2019 p.479-483

Investigation of sperm telomere length as a potential marker of paternal genome integrity and semen quality

Recent studies have reported shorter sperm telomere length (STL) in men with idiopathic infertility. The aim of this study was to measure STL in semen samples from men to evaluate whether STL variation is associated with chromosomal abnormality, DNA fragmentation, traditional semen parameters, IVF outcome, or all four factors. A significant correlation between telomere length and diploidy was observed (P = 0.037). Additionally, STL was found to be positively associated with sperm count (P = 0.006); oligospermic samples had particularly short telomeres (0.9 ± 0.1 versus 1.4 ± 0.1; P = 0.0019). The results confirmed a link between sperm DNA fragmentation and aneuploidy, previously proposed (P = 0.009). A negative relationship was demonstrated between sperm concentration and aneuploidy and Sperm DNA framentation (P = 0.03, P < 0.0001, respectively). For a subset of 51 of the 73 sperm samples used for fertilization, IVF outcomes were known. A total of 17.6% of these samples had atypical STLs. None of these samples produced an ongoing pregnancy. In contrast, the pregnancy rate for samples that had STLs in the normal range was 35.7% (P = 0.044). In conclusion, STL has potential as a fast and inexpensive form of sperm quality assessment.

A study of variation in the levels of seminal plasma superoxide dismutase and glutathione peroxidase in normospermic and oligozoospermic men

Background: Infertility is one of the major concerns of humankind and the male cause of infertility contributes nearly a third for its condition. Amongst many a factor that influences sperm health, the seminal antioxidants are one among the vital determinants of male fertility. Aim and objective: To estimate and compare the levels of seminal plasma superoxide dismutase (SOD) and glutathione peroxidase (GPx) between normospermic and oligospermic men and to correlate them with the seminal parameters of sperm count, motility and vitality of the seminal samples of the respective population. Methodology: This pilot effort is a hospital –based study with the study population comprising of 10 normospermic men for the control group and 10 oligospermic volunteers for the case group. Superoxide dismutase and glutathione peroxidase were estimated in the seminal plasma to reflect the antioxidant status in the seminal sample. Results: SOD levels in normospermic was 11.19± 1.22 (U/ml of seminal fluid) while in oligospermic was 9.17 ± 1.94 (U/ml of seminal fluid) and GPx level in normospermic was 24.46 ±7.39 (µg of GSH consumed /min/ml of seminal fluid)while in oligospermic was 17.23 ±8.27(µg of GSH consumed/min/ml of seminal fluid). SOD is significantly reduced (P value 0.012 (<0.05)) and GPx reduced with P value 0.054 in oligospermic men, which correlates inversely with the seminal parameters of sperm count, motility and vitality in the oligospermic samples . Conclusion: The decreased levels of SOD and GPx in the seminal plasma of oligospermic men when compared to normospermic population signifies a reduced scavenging machinery in their seminal plasma which would have lead to the decrease in the sperm count due to oxidative stress thus leading to infertility.

Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation

BackgroundSpermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation.MethodsSemen samples, obtained from forty-four patients (23–30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation.ResultsMain results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation.ConclusionsZinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.

Sole Use of Sucrose in Human Sperm Cryopreservation

Glycerol alone or in combination with other additives is one of the most widely used and successful cryoprotectants for human sperm. The glycerol method requires rigorous post thaw sample washing for use in ART and this may lead to low sperm yield from oligospermic samples. In this study the feasibility of the use of sucrose in sperm cryopreservation was explored. Sucrose as cryoprotectant was combined with direct plunging of sample into liquid nitrogen (vitrification) as a freezing method. Sucrose treated sperm from normozoospermic and severly oligozoospermic samples underwent rapid freeze and thaw. Motility and viability were evaluated before freezing (after sucrose equilibration) as well as post freezing (after thaw). The 100 mM concentration of sucrose showed better cryoprotectant features compared to that of higher concentrations (200-1000 mM). Sucrose (100 mM)treated sperm maintained low but acceptable motility (30%) and satisfactory viability (60%) after freezing and thawing. The cryoprotectant capacity of sucrose for normozoospermic and oligozoospermic samples were identical. The sucrose method utilizes rapid freezing of a micro volume of sample and thus quickly freezes, thaws, and maximizes recovery of the sperm from the sample.

Cytogenetic Analysis of Sperm Nucleous Components of Iranian Normal and Sub-Fertile Individuals Using Zona Free Hamster Oocytes

The purpose of this study was to investigate whether infertility is affected by sperm chromatin and cytogenetic abnormalities. To this purpose, the frequency of sperm premature chromosome condensation (PCC) induction and numerical chromosome abnormalities in the sperm of normal and sub-fertile men were analyzed. PCC rate was studied for evaluating the role of sperm chromatin abnormalities in the process of nuclear decondensation. Controlled prospective study. Infertility Genetics Department, Royan Institute. Sub-fertile males who were referred for infertility treatment and sperm cytogenetical studies. Hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. Following treatment with Hyaloronidase, zona was removed by trypsin digestion. Sperms were classified according to the morphology, movement and counts and then processed by swim up method. After capacitation, zona-free oocytes were incubated with sperms, and then transferred to fresh media containing colcemid. Slides were prepared using Tarkowskie's standard air-drying technique. Oocytes were analyzed using x 1000 microscope after staining in 5% of Giemsa. The incidence of sperm aneuploidy, PCC and penetration rate in three groups were determined. Regarding the PCC rate, a significantly higher frequency was found in infertile patients. (P < 0.001). The frequency of PCC in oligosperm samples was 36% compared to 19.37% in normal group. A higher frequency of numerical chromosome abnormalities was found in infertile patients. The rate of these abnormalities was 5.6% in normal group and 18.5% in oligospermic samples. Despite the considerable difference between those frequencies, this difference is not significant. (P > 0.05). From the results it can be concluded that, formation of sperm PCC is a major cause of failed fertilization in individuals with sperm abnormalities. PCC may form due to chromatin abnormalities, improper DNA packing, chromosomal abnormalities and penetration delay of sperm. Also this may be involved in the etiology of some cases of idiopathic infertility. About numerical chromosome abnormalities although the differences are not significant, there is an association between sperm numerical chromosome abnormalities and male infertility. These abnormalities can be originated from meiotic process in spermatogenesis.

Ultra-rapid freezing of severe oligospermic samples using cryoloops

Objective: To determine feasibility of ultra-rapid freezing oligospermic samples using cryoloops. Design: Laboratory study Materials/Methods: Sperm motility following exposures to different freezing solutions (F1 and F2) diluted to 4 separate concentrations with test yolk buffer (TYB) was assessed (N=10). Four solutions were further evaluated for changes in motility over 4 min. The two optimal solutions were evaluated as cryoprotectants (C1, C2) during ultra-rapid freezing. Aliquots from 14 semen samples were mixed with C1, C2, C3 (TYB + 12% (v/v) glycerol), and C4 (seminal plasma), placed on cryoloops and directly submerged into liquid nitrogen or placed into liquid nitrogen vapor for 5 min prior to submersion. After freezing, sperm were thawed in TYB (C1–4) or seminal plasma (C4) and motility and viability were assessed. Using the optimal cryoprotectant (C3), post-thaw motility was compared between ultra-rapid and slow-rate freezing (N=10) Results: Sperm motility in F1 1:1 dilution was 77% +/− 2.4% (mean +/− S.E.); 3:1 dilution 51% +/− 4.3%; and 4:1 dilution 39% +/− 5.7% while that in F2 1:1 dilution was 41% +/− 6.3%; remaining tested solutions all had motilities <20% (p <0.05 for all dilutions when compared to initial samples). Sperm motility assessed after 4 min were optimal in F1 1:1 dilution (66% +/− 8.1%) and F1 3:1 dilution (49% +/− 4.4%). These solutions were selected as C1 and C2, respectively. Prior to ultra-rapid freezing, mean sperm motility and viability was 54% +/− 3.4% and 66% +/− 3.8%, respectively. Regardless of tested solutions, post-thaw motility of sperm submerged directly into liquid nitrogen was 0%. Of samples initially suspended in liquid nitrogen vapor, post-thaw sperm motility was 5% +/− 1.9% (C1); 0% (C2); 40% +/− 3.2% (C3); 16% +/− 3.6% and 21% +/− 4.7% (C4 resuspended in seminal plasma or TYB, respectively). Viability of thawed sperm was 4% +/− 2.0% (C1); 0% (C2); 17% +/− 3.9% and 21% +/− 4.7% (C4 resuspended in seminal plasma or TYB, respectively). Viability stain could not be performed on sperm frozen in C3 because of reagent incompatibility. Post-thaw sperm motility of semen frozen with C3 by ultra-rapid means (45 +/− 3.4%) and conventional means (45 +/− 3.1%) were similar (p = 0.95) but less than unfrozen samples (55 +/− 22%; p <0.05) Conclusions: Ultra-rapid freezing of small numbers of sperm (∼100) using cryoloops is feasible. Of cryoprotectants tested, the best post-thaw motility is seen when freezing in TYB + 12% (v/v) glycerol and thawing in TYB alone. Initial post-thaw sperm motility after ultra-rapid freezing with this cryoprotectant is comparable with conventional slow-rate freezing. Considering the comparable results, one can appreciate the potential impact of a technique that takes 5 min compared to conventional slow-rate freezing which takes approximately 1.5 hours. Supported by: None.

Influence of swim-up method on the recovery of spermatozoa from different types of semen samples.

To compare the effectiveness of swim-up method, using different types of semen samples. In this retrospective study undertaken in university medical college infertility centre, subfertile couples undergoing Intra Uterine Insemination were selected. A total of 600 semen samples used for the preparation of sperm samples using swim-up method were analyzed. Relative Yield was calculated from the sperm count and motility before and after swim up from each semen sample in six different groups. Statistically significant increase in relative yield was found in oligospermic samples (20.41) followed by teratospermia (16.98). However, relative yield was low in asthenospermic (11.97) and normal (> 60 million/ml) samples (11.66). Semen samples with good sperm count resulted in poor recovery after swim up. Swim-up method appears to be effective for oligospermic samples. Modifications like multiple tube swim up, using appropriate incubation time based on the initial semen parameter, will enhance the sperm yield after swim up.

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.

Autosomal aneuploidy rate in spermatozoa: a comparison between normospermic and oligospermic males, using a double-color FISH

Aneuploidy rate for chromosomes 3, 7 and 12 in spermatozoa from normospermic (> 20M/ml) and from oligospermic samples (≤ 20M/ml) have been studied with a double-color fluorescence in situ hybridization. In the normospermic samples, the disomy rate for chromosomes 3 and 12 were similar, with the same mean of 0.17%. The disomy frequency for chromosome 7 was significantly higher (z = 2.4, p<0-.05) with a mean of 0.28 %. In the oligospermic samples, there was no significant difference in the disomy rate for chromosomes 3, 7 and 12 (0.24 %; 0.24 %; 0.28 %). Comparing the two semen populations, the rate of disomy 7 was higher in normospermic males while the rate of disomy 12 was significantly higher in oligospermic males. The frequency of diploid sperm was not significantly different between the two groups. Work is in progress to compare moderate and severe oligospermic patients.

Optimal Dose and Duration of Exposure to Artificial Stimulants in Cryopreserved Human Spermatozoa

Purpose Poor sperm motility after cryopreservation is associated with infertility. For any pharmacological stimulation to be of clinical value, its effect in enhancing motility and other motion characteristics should be maintained for at least 1 hour. Materials and Methods Three motility stimulants (pentoxifylline, caffeine and 2-deoxyadenosine) were incubated with post-thaw semen samples from 11 healthy donors for 0, 30, 60, 120 and 180 minutes. The final concentrations used were 2.5 mM., 5 mM. and 10 mM. for pentoxifylline; 1 mM., 2 mM. and 5 mM. for caffeine, and 0.5 mM., 1 mM. and 2.5 mM. for 2-deoxyadenosine. Percent motility and changes in motion characteristics were measured on a computer assisted semen analyzer. Results Compared to controls (0 minutes, no stimulant), an immediate increase in motility and other motion parameters was noted with all 3 stimulants. All stimulants caused a significant increase in percentage motility at all periods studied (p less than 0.01). Similarly, pentoxifylline increased other motion parameters at the 2.5 mM. concentration (p less than 0.01), caffeine was effective in increasing curvilinear velocity, average path velocity and amplitude of lateral head displacement (p less than 0.01) at 5 mM., and 1 and 2.5 mM. of 2-deoxyadenosine increased the curvilinear velocity, straight line velocity, average path velocity and amplitude of lateral head displacement (p less than 0.01). Among all stimulants only 2-deoxyadenosine increased linearity only at the 1 mM. concentration. Conclusions Our study suggests that these stimulants, when used at optimum concentrations, can maintain the improved sperm quality for durations longer than the minimum needed for fertilization. This finding may be significant in improving the poor semen quality observed after cryopreservation in oligospermic samples and in semen specimens from cancer patients.

Protein differences between normal and oligospermic human sperm demonstrated by two-dimensional gel electrophoresis

Protein expression by sperm obtained from men with normal semen analysis and men with oligospermia were evaluated by two-dimensional gel electrophoresis. Proteins were solubilized in a 9.5 M urea/2% Nonidet-P40 (LKB, Bromma, Sweden) lysis buffer and underwent second dimension separation on 10 to 16% polyacrylamide gradient gels. A set of 36 invariant proteins was identified in all normospermic samples, whereas 8 of 10 evaluable oligospermic samples lacked 1 or more of the invariant proteins. Proteins absent in oligospermic samples may be critical to normal sperm function and may serve as markers for infertility.

The fertilization of human oocytes by spermatozoa from men with antispermatozoal antibodies in semen.

Seventy-two couples, including 15 with antispermatozoal antibodies in the male partner's semen, were studied in a program of in vitro fertilization and embryo transfer. Cases were further subclassified as normospermic or oligospermic and antispermatozoal antibodies were assessed with categorization into the respective human immunoglobulin classes as determined using the indirect immunobead test. The study reveals that fertilization is significantly reduced (P less than 0.001) only if both IgA and IgG antibodies are present in semen but there is no reduction if either class is present alone. The fertilization rate of oocytes is significantly reduced (P less than 0.001) by sperm from oligospermic samples, and there is a further reduction in those cases with combined IgA/IgG antispermatozoal antibodies.

The limitations of in vitro fertilization from males with severe oligospermia and abnormal sperm morphology.

Thirty-one patients whose infertility was attributed to oligospermia were included for treatment by in vitro fertilization and embryo transfer. Three subgroups were defined: severe oligospermia (less than or equal to 5 million motile sperm/ml), moderate oligospermia (6 to less than 12 million motile sperm/ml), and abnormal sperm morphology (greater than 60% atypical). The fertilization rates were compared to those of a normospermic group managed concurrently. A modified overlay technique of sperm preparation is described for oligospermic samples so that the number of motile spermatozoa inseminated into each tube or culture dish containing a mature preovulatory oocyte was similar in each category, within the range 0.5 to 2 X 10(5)/ml. Significantly fewer oocytes were fertilized in the severe oligospermic group (P less than 0.001), suggesting a reduced capacity for fertilization by spermatozoa from severely oligospermic males. The fertilization rate of oocytes was normal in the moderate oligospermic group and those with abnormal morphology, although in the latter there was a significant delay noted in reaching the pronuclear stage (P less than 0.001), and the embryos were at a less advanced stage of cleavage at the time of transfer (0.001 less than P less than 0.01). Pregnancies were achieved in both the severe and the moderate oligospermic groups, with healthy infants delivered from each.

Relationships of seminal plasma prolactin with spermatozoal characteristics and fertilizing capacity in vitro.

Seminal plasma prolactin (PRL) levels were determined in 224 semen samples collected from fertile and suspected infertile men, and their relationships with spermatozoal characteristics and fertilizing capacity in vitro were evaluated. PRL concentrations were similar in normospermic and oligospermic samples, but were significantly lower in azoospermic samples. PRL concentrations were significantly higher in samples with high spermatozoal motility than in those with low spermatozoal motility. There were no significant differences in PRL concentrations between normal-morphology and abnormal-morphology samples, and between high-fertilizing capacity and low-fertilizing capacity samples. A low degree of positive correlation was observed between the PRL concentration and the spermatozoal motility. There were no significant correlations between the PRL levels, the other spermatozoal characteristics, and the spermatozoal fertilizing capacity in vitro. These findings indicate that the seminal plasma PRL levels are significantly depressed in azoospermic subjects and suggest that the seminal plasma PRL may play a role in the maintenance of spermatozoal motility. Whether there is a potential function of prolactin in semen on spermatozoal fertilizing capacity, however, cannot be inferred from the present study.