Biomedical subjects

D R Franken

Publications and source records attributed to D R Franken.

At least 19 recordsLinked to original sources

PubMed2002

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding.

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.

Female↗

PubMed2002

Zona pellucida induced acrosome reaction, sperm morphology, and sperm-zona binding assessments among subfertile men.

PURPOSE: The study aimed to evaluate the relationship between the zona pellucida induced acrosome reaction (ZIAR) and (i) percentage normal spermatozoa as well as (ii) sperm-zona pellucida binding potential among men referred for a routine semen analysis. METHODS: Semen samples of 164 consecutive men referred to the andrology laboratory for routine semen analysis were studied. Semen samples were analyzed using the new WHO standards (strict criteria). ZIAR was recorded with a lectin conjugated Pisum sativum agglutinin microassay, while sperm-zona binding was evaluated with a standard hemizona assay (HZA). RESULTS: Andrology patients were divided according to the percentage normal spermatozoa in the ejaculate, namely <4% normal forms (n = 71), 5-14%, normal forms (n = 73), and >14% normal forms (n = 20). ZIAR data of the <4%, 5-14%, and >14% groups was (9.6 +/- 0.6)%, (13.9 +/- 0.5)%, and (15.0 +/- 1.1)%, respectively. The ZIAR data of fertile control men was (26.6 +/- 1.4)% which differed significantly from the three andrology referrals groups. Likewise significant differences were recorded during the hemizona assay namely, 38.0% (<4% normal forms), 54.5% (5-1% normal forms), and 62.6% (>14% normal forms). Among the group with >14% normal forms, five cases had impaired ZIAR outcome (<15%). Three of these men had normal morphology and HZAs. CONCLUSIONS: ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of andrology referrals revealed an impaired acrosome reaction response to solubilized zona pellucida.

Acrosome Reaction↗

PubMed2002

Defective sperm decondensation: a cause for fertilization failure.

The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups, < 44%, n = 10; > or = 44-59%, n = 10; and > or = 60%, n = 29. Morphology groups were < or = 4% (n = 11); > 4-14% (n = 19); and > 4% (n = 19). One hundred and seventy-two oocytes that failed IVF were evaluated for sperm-zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the > or = 60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3, staining of < 44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with > 4-14% normal cells, while it increased 2.45 fold for the morphology group with < or = 4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups > or = 44-59% and < 44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.

Chromatin↗

PubMed2002

Extracellular signal-regulated kinase activation involved in human sperm-zona pellucida binding.

In a previous study involving the inhibition the mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), we found that the very specific MAPK kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced acrosome reaction. As an intact acrosome on the spermatozoa is a prerequisite in ensuring tight binding to the ZP, we investigated the zona binding potential of spermatozoa after PD098059 treatment of sperm, followed by exposure to solubilised human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa, exposed to solubilised ZP, bound significantly more to the ZP, as compared to control spermatozoa also exposed to solubilised ZP (26.5 +/- 3.7 vs. 13.8 +/- 2.8, P < 0.05). No significant differences in binding to the ZP were observed between PD098059 treated and untreated sperm populations after A23187 exposure. These results can be interpreted to support the idea that the ZP-induced AR is the physiologically relevant exocytotic event, as it is the ZP-induced AR, and not the spontaneous (culture medium) or A23187 induced AR, that appears to be mediated through an ERK-mediated signal transduction process.

Calcimycin↗

PubMed2001

Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida.

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.

Acrosome Reaction↗

PubMed2001

Development of a new, highly sensitive zona pellucida binding assay using a bioluminescence-enhanced detection system.

To date, two different zona binding assays have been described in the literature. Both assays, however, require a large quantity of human zonae which vary immensely in quality. Furthermore, an inverted microscope with micromanipulation equipment is necessary, which makes both assays relatively complicated and time-consuming, and requires skilled staff. Therefore, we developed a new, highly sensitive zona binding assay using a bioluminescence-enhanced system which employs a pool of solubilized zona pellucida and is easier for routine use. In the detection system, light emission by the luciferin-luciferase system is measured. Because of the limited availability of human zonae pellucidae, this new assay was first developed in the porcine system. The new bioluminogenic substrate D-luciferin-O-beta-galactopyranoside (Lu-Gal) was synthesized, purified and characterized. Synthesis of Lu-Gal resulted in purity better than 99.998%. Analytical data and spectra were appropriate. In terms of the kinetic data, Lu-Gal is a highly sensitive and specific substrate for beta-galactosidase. Using the given chemical conditions, nonlabelled zonae bound competitively to boar spermatozoa, which resulted in a high sensitivity and specificity. By the addition of 10 nonlabelled zonae, the binding of labelled zonae was almost completely inhibited. Corresponding results were obtained when the bioluminescent system was compared with the hemizona assay. On the other hand, spermatozoa of other species (bull, hamster and man) showed only low binding to the porcine zonae or none at all. Competitive displacement was not observed, indicating the inter-species specificity of the assay.

Animals↗

PubMed2001

The zona pellucida-induced acrosome reaction of human spermatozoa involves extracellular signal-regulated kinase activation.

Extracellular signal-regulated kinases (ERKs), belonging to the family of mitogen-activated protein kinases (MAPKs), are cytoplasmic and nuclear serine/threonine kinases involved in the signal transduction of several extracellular effectors. Recent evidence indicates the presence of p21 Ras and the phosphorylation of ERK1 and ERK2, suggesting the occurrence of the Ras/ERK cascade in mammalian spermatozoa. The present article describes the biological role of ERK during the acrosome reaction of human spermatozoa on stimulation with zona pellucida (ZP). The mitogen-activated protein-kinase inhibitor PD098059 was used as a pharmacological tool to study the involvement of extracellular signal-regulated kinases in the induction of the acrosome reaction in human spermatozoa. This compound significantly inhibited the acrosome reaction induced by both ZP and the calcium ionophore A23187. These results suggest that ERKs are involved in the signal transduction pathway through which ZP stimulation works during the process of fertilization.

Acrosome↗

PubMed2001

Clinical importance of zona pellucida-induced acrosome reaction and its predictive value for IVF.

The study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. ZIAR tests were performed using 0.25 zona pellucida/microliter co-incubated with spermatozoa for 60 min. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between stimulated and unstimulated (spontaneous) sperm populations. Results were compared with IVF rates of metaphase II oocytes. Interactive dot diagrams divided the patients into two groups, i.e. ZIAR <15% and ZIAR > 15%, with mean fertilization rates of 49 and 79% respectively. The sensitivity and specificity for ZIAR results versus fertilization were 93 and 100% respectively. The area under the curve was 99% and the 95% confidence interval did not include 0.5 which implies that the ZIAR test is able to predict fertilization failure among IVF patients. In conclusion, the ZIAR test has diagnostic potential since it can assist the clinician to identify couples that will benefit from intracytoplasmic sperm injection therapy.

Acrosome Reaction↗

PubMed2000

A continuous quality control program for strict sperm morphology.

OBJECTIVE: To develop a training program with intervals of continuous quality control assessments for the evaluation of strict sperm morphology. DESIGN: Prospective analytical study. SETTING: Academic hospital and academic institution setting. PATIENT(S): Healthy sperm donors. INTERVENTION(S): Nine individual andrology laboratories in Switzerland were invited to participate in a training course for strict sperm morphology, which was followed up every 3 months by a continuous quality control program. Each laboratory received six slides over a period of 9 months, during which time the results were forwarded to the reference laboratory. Papanicolaou stain sperm slides were prepared and shipped to participating laboratories every 3 months. MAIN OUTCOME MEASURE(S): Percentage of normal spermatozoa. RESULT(S): The mean (+/-SE) percentage of normal sperm reported by the reference laboratory compared with the participating laboratories for slides 1-6 were 11. 4 +/- 1.6 vs. 17.3 +/- 6 (P>.2), 6.0 +/- 1.3 vs. 8.6 +/- 2.5 (P>.2), 9.0 +/- 0 vs. 9.6 +/- 3 (P>.2), 1 +/- 0 vs. 1.2 +/- 0.2 (P>.2), 23.3 +/- 0.3 vs. 28.0 +/- 1.3 (P>.2), and 2.0 +/- 0 vs. 6.1 +/- 1.2 (P>.2), respectively. Technician proficiency was reported to differ by <10% from the reference laboratory in 94% of cases. CONCLUSION(S): The results illustrate that training and proficiency testing can be conducted on a national and international level with the support of a reference laboratory. Global quality control measurements in andrology laboratories should become mandatory, since these results indicate that continuous quality control for laboratory technicians can be successful.

Humans↗

PubMed2000

Physiological induction of the acrosome reaction in human sperm: validation of a microassay using minimal volumes of solubilized, homologous zona pellucida.

PURPOSE: The objective was to develop a method that could accommodate microvolumes of solubilized human (ZP) and sperm for assessing the induction of the acrosome reaction. METHODS: A microassay using 1 microliter of 2.5, 1.25, 0.6, 0.3, and 0.125 ZP/microliter incubated with 1 microliter of a highly motile sperm suspension for 60 min. As a control and parallel to the microassay a standard acrosome reaction technique was performed. RESULTS: No significant differences were observed between the percentage acrosome-reacted sperm reported by the two assays under basal conditions (spontaneous) or after induction with a Ca2+ ionophore or solubilized ZP. At a ZP concentration of 0.6 ZP/microliter, the percentages of acrosome-reacted spermatozoa in both techniques were significantly higher compared to the spontaneous acrosome reaction results, namely 18% and 17%, compared to 10% and 10%, respectively. Approximately a 30% level of acrosomal exocytosis was induced with 2.5 ZP/microliter both methods. CONCLUSIONS: This newly devised microtechnique is easy and rapid to perform, is repeatable, and facilitates the use of minimal volumes of solubilized human ZP (even a single ZP) for assessment of the inducibility of the acrosome reaction of a homologous sperm population.

Acrosome Reaction↗

PubMed2000

Physiological induction of the acrosome reaction in human sperm: validation of a microassay using minimal volumes of solubilized, homologous zona pellucida.

PURPOSE: To develop a method that could accommodate microvolumes of solubilized human zona pellucida (ZP) and sperm for assessing the induction of the acrosome reaction. METHODS: A microassay using 1 microliter of 2.5, 1.25, 0.6, 0.3, and 0.125 ZP/microliter incubated with 1 microliter of a highly motile sperm suspension for 60 min. As a control and parallel to the microassay a standard acrosome reaction technique was performed. RESULTS: No significant differences were observed between the percentage acrosome reacted sperm reported by the two assays under basal conditions (spontaneous) or after induction with a Ca2+ ionophore or solubilized ZP. At a ZP concentration of 0.6 ZP/microliter, the percentages of acrosome-reacted spermatozoa in both techniques were significantly higher compared to the spontaneous acrosome reaction results, namely, 18% and 17%, compared to 10% and 10%, respectively. An approximately 30% level of acrosomal exocytosis was induced with 2.5 ZP/microliter in both methods. CONCLUSIONS: This newly devised microtechnique is easy and rapid to perform, is repeatable and facilitates the use of minimal volumes of solubilized human ZP (even a single ZP) for assessment of the inducibility of the acrosome reaction of a homologous sperm population.

Acrosome Reaction↗

PubMed2000

Chromatin packaging as an indicator of human sperm dysfunction.

PURPOSE: Understanding the causes of fertilization failure is an important research field in assisted reproductive programs. The present study aimed to evaluated the possible relationship between chromatin packaging quality (CMA3 staining) and (i) normal morphology and (ii) its ability to predict the functional integrity of spermatozoa in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment programs. METHODS: Semen of 140 men from IVF and ICSI couples were analyzed for sperm concentration, motility, morphology, and chromatin packaging (CMA3). For CMA3 classification, two cutoff values were used, namely, 44.5% +/- 13 and 1 SD above the mean, i.e., 57.5% (rounded off to 60%). IVF and ICSI data were stratified using three basic cutoff values for CMA3 staining, namely, < 44%, > 44-60%, and > 60%. RESULTS: Based on CMA3 results patients were divided into four groups, namely, group A, < 44% CMA3 (n = 15, IVF); group B, > or = 44% and < 60% CMA3 (n = 39, IVF); group C, > or = 60% CMA3 (n = 45 IVF); and group D, > or = 60% CMA3 (n = 41 ICSI). During receiver operator characteristic analyses the estimated cutoff value for CMA3 staining, to distinguish between < 4% and > or = 4% morphology groups, was 60%. The area under the curve was 0.89, sensitivity of 75%, and specificity of 100%. When IVF rates of > 60% and < 60% were used, the optimal CMA3 value for prediction of fertilization success again was recorded at 60%. The area under the curve was 0.76, sensitivity of 81.5%, and specificity of 63.6%. CONCLUSIONS: Chromatin packaging assessments should be included as a complementary assay to the sequential diagnostic approach of the male-factor patients.

Chromatin↗

PubMed2000

Sperm chromatin packaging as an indicator of in-vitro fertilization rates.

The development of a sequential diagnostic schedule for patients consulting for infertility disturbances would be an ideal method of approach for clinicians in the absence of an aetiological or pathophysiological diagnosis. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology as well as in-vitro fertilization (IVF) rates, with chromatin staining among fertile and subfertile men. Two semen smears were prepared from each specimen obtained from 72 men to record normal morphology and chromatin packaging as recorded by chromomycin A(3) (CMA(3)) staining. Following the semen analyses, the 72 men were divided into the two morphological groups, namely <4% and >4% normal forms. Significantly different percentages of CMA(3) staining (mean +/- SE) were recorded between the two morphological groups, namely 65.9% +/- 3.5 and 44.5% +/- 1.7 (P +/- 0.001). A highly negative significant correlation existed between percentage of normal morphology as recorded by strict criteria and CMA(3) staining. A highly significant and positive correlation was recorded for normal morphology and IVF rates (r +/- 0.45, P +/- 0.0001). A significant negative correlation (r +/- -0.51, P +/- 0.0001) existed between CMA(3) values and IVF rates. The discriminating power of nuclear maturity, as recorded by CMA(3) staining, to identify abnormal morphology values and poor IVF rates was calculated with receiver operator characteristic (ROC) analyses. The areas under the ROC curves were 0.86 for sperm morphology and 0. 74 for IVF rates. The calculated threshold values for CMA(3) staining to distinguish between morphology groups were 48 and 50% for IVF. Chromatin packaging assessment is a valuable addition to the sequential diagnostic programme in an assisted reproductive arena.

Chromatin↗

PubMed2000

The development of a continuous quality control programme for strict sperm morphology among sub-Saharan African laboratories.

Inter-technician and between-laboratory differences, especially during the evaluation of sperm morphology, have been a major cause of concern. The study aimed to develop an intensive training programme with intervals of continuous quality control assessments for sperm morphology. Twenty andrology laboratories from sub-Saharan Africa were invited to participate in a World Health Organization Special Programme of Research, Development and Research Training in Human Reproduction semenology workshop. Following intensive training in strict sperm morphololgy evaluation, a continuous quality control programme was introduced on a quarterly basis. At baseline, the mean (+/- SD) percentage difference reported between the participants and the reference laboratory reading was 33.50 +/- 11%. After training, the mean percentage difference had decreased to 14.32 +/- 5% at 3 months and to 5.00 +/- 5% at 6 months. Pairwise comparison of the differences at each evaluation time revealed the following: Baseline differences (pre-training) differed significantly from the differences at 3 months (P = 0.0002) as well as at 6 months after training (P = 0.007). The differences at 6 months did not differ significantly from those at 3 months (P = 0.27). Training of andrology technicians as well as continuous proficiency testing can be conducted on a national and international level with the support of a referring laboratory. Global quality control measurements in andrology laboratories should become mandatory, since these results indicate that continuous quality control for laboratory technicians can be highly successful.

Africa, Northern↗

PubMed1999

Use of failed-fertilized oocytes for diagnostic zona binding purposes after sperm binding improvement with a modified medium.

PURPOSE: Because the availability of prophase oocytes for zona binding testing is limited, we compared sperm binding to the zona of failed-fertilized intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) oocytes after incubation in a standard IVF medium and a specially composed binding improvement medium. METHODS: Semen samples from nine patients and nine fertile donors were separated in parallel by the standard swim-up method in both media. Subsequently, hemizona assays were performed with prophase, failed-fertilized ICSI and IVF oocytes. RESULTS: Sperm separation resulted in a significantly higher sperm count (P < 0.01) and progressive motility (P = 0.018) in binding improvement medium. Moreover, spermatozoa coincubated with hemizonae (prophase, failed-fertilized ICSI and IVF oocytes) in binding improvement medium bound significantly more to hemizonae than in the controls (P < 0.01). However, the hemizona index did not differ. CONCLUSIONS: Thus, the limited number of human zonae can be increased by the use of oocytes that failed to fertilize during ICSI or IVF. This will lead to a qualitative improvement of the diagnostic spectrum in male-factor infertility.

Adolescent↗

PubMed1999

Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining.

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.

Aniline Compounds↗

PubMed1999

Characterization of human zona pellucida glycoproteins.

The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy.

Acrosome Reaction↗

PubMed1998

The clinical significance of sperm-zona pellucida binding.

The development of homologous functional bio-assay for sperm quality assessment has been a focal point of reproductive biologists; in order to provide a scientific based diagnosis in cases of fertilization failure. The availability of oocytes still remains an important limiting factor for laboratories to embark on the methodology of the assay. The use of zonae pellucidae, derived from post mortem and different in vitro fertilization oocytes, enhanced to availability of zonae. Sperm-zona binding has been illustrated to be an essential requisite during human fertilization and can be measured under hemizona assay as well intact zona pellucida conditions. The sensitivity and specificity of sperm-zona binding results indicated the assay to be positively and significantly correlated with in vitro fertilization outcome. Furthermore, a highly significant correlation were illustrated to exist between the normal sperm morphology, hyperactivation, sperm creatine kinase activity and the zona binding capacity of a given sperm sample. It was concluded that andrology testing remains an ever-growing component in the work-up of the infertile couple. We enter the next millennium with many questions that remain to be answered by the hand of efficacious screening techniques and a new formidable therapy in intra cellular sperm injection.

Female↗