Sub-zonal insemination for extreme male factor infertility

When in-vitro fertilization (IVF) is used for severe male infertility,the zona pellucida constitutes a major barrier to sperm —oocyte interaction, a barrier that may, in principle, be overcomeby micro-injecting one or more spermatozoa into the sub-zonalperivitelline space (‘sub-zonal insemination’ orSZI). We have defined suitable patients for SZI as having ‘extreme’male factor in that they have either shown a failure of fertilizationin previous IVF cycles or had < 50 000 motile spermatozoarecoverable after semen preparation. (This is distinct fromthose with only ‘severe’ male factor in whom sufficient(> 50 000) motile spermatozoa could be recovered from a semenpreparation.) A total of 213 SZI cycles were performed at SydneyIVF in the 4 year period September 1988 to September 1992, forextreme male factor patients with previous IVF failures or extremelylow sperm numbers for whom SZI was the first option (about two-thirdsand one-third of cases respectively). A total of 138 embryotransfers are reported, producing 20 clinical pregnancies afterperforming SZI on 1899 oocytes. One patient miscarried at 12weeks gestation and there have been nine normal deliveries (sofar) of 10 healthy infants. The first delivery was in February1990. One pregnancy was achieved in the only patient in whomspermatozoa were obtained by epididymal aspiration, and transferof three cryopreserved embryos in another patient resulted ina singleton pregnancy. Of the 492 oocytes fertilized, 282 hadtwo pronuclei (57.3%) and normal embryos were transferred in138/213 (64.8%) treatment cycles, giving an overall pregnancyrate of 14.5% per embryo transfer or 9.4% per cycle. These resultsare considerably better than those obtained in a subpopulationwho had also undergone IVF (average fertilization rate = 4.2%,no pregnancies), although lower than traditional IVF in coupleswith severe male factor. This emphasizes that selecting an appropriatepatient population for SZI is critical in establishing its trueclinical relevance. The major limitation to the technique isthe need for spermatozoa to be acrosome-reacted before injection.     

Ubiquitin-based sperm assay for the diagnosis of male factor infertility

Sperm morphology is correlated with fertility in men, yet the existing, subjective sperm morphology assays provide only a limited insight into patients' infertility. Here, we provide the experimental background for a new, objective and automated semen assay, based on the cross-reactivity of defective human spermatozoa with antibodies against a proteolytic marker peptide, ubiquitin. Using immunofluorescence and flow cytometry, we screened the spermatozoa from 17 infertility patients and two fertile donors for their cross-reactivity with anti-ubiquitin antibodies. Thirteen out of 17 patients, but neither of two fertile donors, displayed an increased binding of anti-ubiquitin antibodies to sperm surface, that reflected the occurrence of abnormalities in these samples and was corroborated by available clinical data. Highest correlation coefficient (r = -0.432) was obtained with the cleavage rate after IVF. The contribution of male factor was revealed in several couples previously diagnosed with idiopathic infertility. Ubiquitin-cross-reactive sperm-surface proteins thus seem to be a universal marker of semen abnormalities, including sperm head and tail defects and semen contaminants such as spermatids, leukocytes and cellular debris. We propose that the sperm-ubiquitin tag immunoassay (SUTI) may be a valuable diagnostic tool in treatment of male factor and idiopathic infertility.     

Can we justify spermatid microinjection for severe male factor infertility?

During 1995 and 1996 the first spermatid pregnancies were announcedwith both round spermatid ROSI) and elongated spermatid ELSI)injections. These publications were flanked by live births fromROSI in a number of animal species, with resulting offspringappearing normal, healthy and fertile. However, the live birthsin humans heralded a scientific and ethical debate on the clinicaluse of this technology; and in a number of countries nationwidemoratoria prohibiting spermatid microinjection were enjoined.Concerns surrounded the biological condition of spermatids andclinical implications of utilizing an immature spermatozoanfor conception. Nevertheless, case reports and a few scientificstudies on human spermatid conception have been published inrecent years, and further polemic on testicular histopathologyand prognosis has ensued. This paper reviews the current argumentson the clinical use of ROSI and ELSI, and evaluates the biologyof the main contributory components of a spermatozoan to thesubsequent embryo, namely the genetic material, the microtubularorganizing complex and the putative oocyte activating factor.We also consider the relevant testicular histopathology andlikely outcome in the context of the current birth rate fromROSI and ICSI. We conclude by considering the way forward forinfertile men who require this technology to become geneticfathers, and whether the time is now appropriate to considerclinical trials.     

Routine application of partial zona dissection for male factor infertility

Partial zona dissection (PZD) increases the chances of fertilization by improving the access of spermatozoa to the perivitelline space (PVS) helping those spermatozoa unable to penetrate the zona pellucida (ZP) and possibly those poorly able to penetrate the oolemma. Problems arise in assessing semen to decide which parameters might indicate defects of this nature. PZD, by circumventing the ZP, may also increase the rate of polyspermy, especially in infertility where ZP and oolemmal penetration are not defective. Given these drawbacks, we performed PZD as routine treatment for male infertility in 70 in-vitro fertilization cycles. In three different groups, PZD proved to be either effective, ineffective or unnecessary. In the first group of 35 cycles, fertilization was 23% with initial PZD and 33% with PZD reinsemination (36% and 41% polyspermy respectively). No fertilization occurred following conventional insemination (CONV). Four pregnancies occurred in this group. In a second group of 19 cycles, fertilization did not occur with either PZD or CONV. In the final group of 16 cycles, fertilization was similar following both PZD and CONV, but polyspermy was 48% in the PZD category. Transfer of mixed PZD and CONV embryos in this group yielded 10 pregnancies. Assessment of all patient and seminal profiles, and those in an oligozoospermic subcategory, revealed no parameters of relevance to success or failure with PZD. However, one subgroup in the group of total failure to fertilize did have a significantly lower percentage of normal morphology (P less than 0.005), suggesting that degree of teratozoospermia may be a prognosticator of success using PZD.     

Seminal plasma alpha-glucosidase activity and male infertility

Measurement of alpha-glucosidase (alpha-GLUC) activity by means of a simple colorimetric test using a commercial kit (EpiScreen; FertiPro, Lotenhulle, Belgium) yielded results that were strongly correlated with the values obtained for the neutral iso-enzyme measured by a fluorimetric reference method (r=0.85, P=0.003, n=13). The former method was characterized by a low intra- and inter-coefficient of variation (6.6 and 4.3% respectively). Vasectomized men with azoospermia (n=27) had a significantly lower alpha-GLUC activity in semen than vasectomized men with residual spermatozoa present (n=11, P < 0.01) and men with azoospermia of primary testicular origin (n=33, P < 0.01). Receiver operating curve (ROC) analysis showed alpha-GLUC measurement to be reasonably accurate in differentiation between cases with obstructive versus testicular azoospermia at criterion value 13.5 U/l (sensitivity=82%, specificity= 70%). In cases with spermatozoa present, alpha-GLUC activity and output per ejaculate were positively correlated with sperm concentration (r=0.53 and 0.38, n=472), linear velocity (r=0.35 and 0.30, n=224), curvilinear velocity (r=0.32 and r=0.29, n=224), semen adenosine triphosphate (r=0.35 and 0.26, n=64), the concentration of 5alpha-dihydrotestosterone (r=0.31 and 0.29, n=74), and gamma-glutamyltransferase activity (r=0.62 and 0.32, n=275) in seminal plasma. The activity of alpha-GLUC was inversely correlated with ROS generation after 12-myristate, 13-acetate phorbol ester stimulation (r=-0.27, n=104), and both alpha-GLUC activity and total output were inversely correlated with the concentration of peroxidase- positive white blood cells among samples with > or =1x10(6)/ml of these cells (r=-0.30 and -0.19, n=165). It is concluded that simple photometric measurement of alpha-GLUC activity in seminal plasma reflects the functional state of the epididymis and may be helpful for the differential diagnosis of certain cases with azoospermia.      

The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility.

The imbalance between reactive oxygen species (ROS) production and total antioxidant capacity (TAC) in seminal fluid indicates oxidative stress and is correlated with male infertility. A composite ROS-TAC score may be more strongly correlated with infertility than ROS or TAC alone. We measured ROS, TAC, and ROS-TAC scores in semen from 127 patients and 24 healthy controls. Of the patients, 56 had varicocele, eight had varicocele with prostatitis, 35 had vasectomy reversals, and 28 had idiopathic infertility. ROS levels were higher among infertile men, especially those with varicocele with prostatitis (mean +/- SE, 3.25 +/- 0.89) and vasectomy reversals (2.65 +/- 1.01). All infertile groups had significantly lower ROS-TAC scores than control. ROS-TAC score identified 80% of patients and was significantly better than ROS at identifying varicocele and idiopathic infertility. The 13 patients whose partners later achieved pregnancies had a mean ROS-TAC score of 47.7 +/- 13.2, similar to controls but significantly higher than the 39 patients who remained infertile (35.8 +/- 15.0; P < 0.01). ROS-TAC score is a novel measure of oxidative stress and is superior to ROS or TAC alone in discriminating between fertile and infertile men. Infertile men with male factor or idiopathic diagnoses had significantly lower ROS-TAC scores than controls, and men with male factor diagnoses that eventually were able to initiate a successful pregnancy had significantly higher ROS-TAC scores than those who failed.     

Immunobiology of male infertility

Experimental and clinical data reported In the literature emphasizethe important role that immune factors may play in the genesisof male infertility even if many problems still remain to besolved. Auto or homo-sensitization in animals (and in male volunteers)can be obtained with testicular homogenate or epididymal spermatozoaand complete Freund's adjuvant. Immune orchitis in spontaneoushuman pathology has also been reported. Vasectomy for the voluntarycontrol of male fertility may be considered a particular formof experimental autoimmunization; and many vasectomized in dividualsdevelop antisperm antibodies in blood serum and/or in seminalplasma. In spontaneous male infertility antisperm antibodiescan: (i) be a mere epiphenomenon; (ii) be a factor aggravatinga pathologic situation already able to cause infertility; (iii)play a pathogenetic role in some forms of so-called Idiophaticinfertility and so could be defmed as infertifity due to antispermantibodies, if the antisperm autoimmane reaction representsthe causal factor of infertility, corticosteroid therapy seemsto give the most satisfactory results, administered either inhigh doses for a very short time period or in low doses overa prolonged period, or even after transient pharmacologicallyinduced azoospermia.     

Alexithymia in male infertility

The continuing confrontation with the thoughts and feelings surrounding an unfulfilled wish for a child makes coping very difficult. There is empirical evidence that, in medical illnesses associated with stress and loss of quality of life, patients react with alexithymia, which means a difficulty to communicate emotions. In this study we compared 84 infertile men with a group of 96 healthy men and 43 male psychosomatic outpatients concerning their ability to communicate feelings, measured by the Twenty-Item Toronto Alexithymia Scale and the amount of psychopathologically relevant symptoms, especially somatization, measured by the Symptom Checklist 90-R and a List of Complaints (Beschwerden-Liste). The results showed a significantly higher alexithymia in infertile men compared with healthy men (P < 0.05), but a significantly lower alexithymia compared with psychosomatic outpatients (P < 0.05). Furthermore the study group showed significantly more somatic complaints in the List of Complaints compared with healthy men (P < 0.05). The importance of alexithymia in male infertility is discussed on the basis of empirical results that it might play a defensive role as far as depression is concerned but on the other hand increases the possibility of somatic complaints. The need for prospective studies in further research is emphasized.     

Sperm treatment with extracellular ATP increases fertilization rates in in-vitro fertilization for male factor infertility

Previous work from our laboratory has revealed that extracellular ATP is a rapid and potent activator of human sperm acrosome reaction and fertilizing ability. In the present study, we assessed the effects of in-vitro sperm incubation with ATP on fertilization and embryo development in couples undergoing in-vitro fertilization (IVF) for male factor infertility. Oocytes from 22 women undergoing ovulation induction were divided in two groups and inseminated in vitro either with selected spermatozoa from the corresponding partner suffering from male factor infertility pre-incubated with ATP (2.5 mM) for 1 h, or with spermatozoa incubated with 0.9% NaCl solution (control group). After insemination, fertilization was assessed by the presence of pronuclei and then by embryo cleavage. The fertilization rate in the group of oocytes inseminated with ATP-treated spermatozoa improved significantly with respect to the control group (65.7 versus 42.5%, P < 0.01). No significant differences were observed in embryo cleavage and embryo quality. Embryos from both treated and control groups were transferred together in 20 transfer procedures, and in two couples fertilization was not obtained. Nine pregnancies occurred: one biochemical, one miscarriage, and seven patients delivered 9 healthy babies. Two pregnancies were twin with an overall pregnancy rate of 40.9% per cycle and of 45% per transfer. In conclusion, the results of the present study demonstrate that, in humans, extracellular ATP induces a significant increase of sperm fertilizing potential, as these findings are a rationale for the use of ATP for in-vitro treatment of human spermatozoa during IVF.     

Secondary male factor infertility after Roux-en-Y gastric bypass for morbid obesity: case report

Surgical treatments such as the Roux-en-Y gastric bypass operation result in the successful treatment of morbid obesity; however, this type of operation may cause long-term side effects due to the reduced absorption of nutrients. Here, we present data suggesting that this operation can result in secondary infertility in males. Six healthy, previously fertile male subjects presented in our centre for secondary infertility after a Roux-en-Y gastric bypass operation for morbid obesity. Reproductive function was assessed with a series of spermiograms, and by testicular biopsy. Secondary azoospermy with complete spermatogenic arrest was diagnosed. The results suggest that weight reduction surgery may cause reproductive dysfunction.     

The impact of treatment experiences on the course of infertility distress in male patients

BACKGROUND: Previous research on infertile males has delivered equivocal findings on the course of infertility distress in males. The present longitudinal study examines whether there are differentials associated with specific treatment experiences (i.e. duration of treatment, the diagnosis received, and treatment failure of assisted reproductive technologies). METHODS: The sample consisted of 118 patients who twice visited an andrology clinic on their own initiative for fertility work-ups. Baseline and follow-up examinations were > or = 6 months apart. Prior to each fertility work-up, patients completed a questionnaire assessing distress due to infertility. RESULTS: No uniform course of distress could be detected. A significant interaction between treatment experiences indicated that distress rises significantly only in those patients who were in treatment > or = 17 months and experienced treatment failure between the first and the second psychological evaluation. For the diagnosis of male infertility, however, neither a direct nor an indirect impact was identified. CONCLUSION: The present study indicates that the interaction of specific treatment experiences is associated with changes in distress of infertile males.     

Risk factors for female and male infertility: results of a case-control study.

In order to evaluate male and female risk factors for infertility in a case-control study, we have compared all couples in a French administrative region consulting for primary or secondary infertility (of more than one year's duration) with couples in which the woman had given birth during the year of the study. For any one couple, a history of varicocele, male genital infections or testis damage multiplies the risk of primary infertility by a factor of 28, 3 and 4 respectively, and previous female infections (salpingitis) multiply the risk by 45. Similarly, a history of ectopic pregnancy, sexually transmitted diseases or salpingitis multiplies the risk of secondary infertility by a factor of 5, 4 and 7 respectively, and a history of varicocele multiplies this risk by 4.     

Infertility: Cancer and male infertility

Male cancer and infertility are linked in three different ways.First, in certain malignancies, such as Hodgkin's disease, testicularand endocrine cancers, infertility may precede the diagnosisof cancer. The cause of infertility in these cases is not alwaysobvious, although endocrine and immune aetiologies are responsiblein some of them. Therefore, during evaluation of an infertilemale, the possibility of cancer should be borne in mind. Second,cryptorchidism causes, in a parallel course, infertility andcancer. Third, improved survival of childhood and adolescentcancer is the trend of modern medicine. However, infertilityis an important major sequela of treatments. The roles of chemotherapy,radiotherapy and surgery on fertility potential are stressedand possible treatment modalities in such cases are discussed.     

Role of caspases in male infertility

Apoptosis is characterized by a variety of changes resultingin the recognition and phagocytosis of apoptotic cells. Caspases(cysteinyl aspartate-specific proteinases) play a central rolein the regulation of apoptosis in the human seminiferous epithelium.They are expressed as inactive proenzymes and participate ina cascade triggered in response to pro-apoptotic signals. Todate, 14 caspases have been implicated in the human apoptoticpathway cascade. Among these, caspase-3 is considered to bea major executioner protease. Since apoptosis is a universalsuicide system in almost all cells, a close control via molecular,endocrine and physical factors establishes homeostasis of cellgrowth and death. The proper regulation of the caspase cascadeplays an important role in sperm differentiation and testicularmaturity. However, caspases have been implicated in the pathogenesisof multiple andrological pathologies such as impaired spermatogenesis,decreased sperm motility and increased levels of sperm DNA fragmentation,testicular torsion, varicocele and immunological infertility.Future research may provide a better understanding of the regulationof caspases, which may help us to manipulate the apoptotic machineryfor therapeutic benefits. In this review, we summarize the consequencesof caspase activation, aiming to clarify their role in the pathogenesisof male infertility.     

Comparison of in-vitro fertilization in male and tubal infertility: a 3 year survey.

The in-vitro fertilization and embryo transfer (IVF-ET) procedure was compared in 175 couples with male infertility and 480 couples with tubal infertility. In cases of male infertility, more oocytes were recovered but fewer oocytes were fertilized. Although the cleavage rate was decreased, no difference in embryo quality was found. In male infertility, fewer transfers were performed and the average number of embryos per transfer was lower. The total pregnancy rate was also lower per cycle (12.8 versus 22.9%), but not per transfer (25.4 versus 25.7%). The further obstetrical outcome was similar in both groups. We conclude that male infertility can be treated by IVF-ET but results are still disappointing when compared to a control group with normal spermatozoa.     

Clinical relevance of oxidative stress in male factor infertility: an update

Male factor has been considered a major contributory factor to infertility. Along with the conventional causes for male infertility such as varicocele, cryptorchidism, infections, obstructive lesions, cystic fibrosis, trauma, and tumors, a new, yet important cause has been identified: oxidative stress. Oxidative stress (OS) is a result of the imbalance between reactive oxygen species (ROS) and antioxidants in the body, which can lead to sperm damage, deformity and eventually male infertility. This involves peroxidative damage to sperm membrane and DNA fragmentation at both nuclear and mitochondrial levels. OS has been implicated as the major etiological factor leading to sperm DNA damage. OS-induced DNA damage can lead to abnormalities in the offspring including childhood cancer and achondroplasia. In this article, we discuss the need of ROS in normal sperm physiology, the mechanism of production of ROS and its pathophysiology in relation to male reproductive system. The benefits of incorporating antioxidants in clinical and experimental settings have been enumerated. We also highlight the emerging concept of utilizing OS as a method of contraception and the potential problems associated with it.     

Klinefelter's syndrome in the male infertility clinic

The clinical features of patients with Klinefelter's syndrome attending a male infertility clinic have been investigated in order to consider their assisted reproduction treatment options. Over 12 years, a total of 148 patients with sterility due to azoospermia had Klinefelter's syndrome. Eight patients were shown by fluorescence in-situ hybridization (FISH) on metaphase spreads to be mosaic (46,XY/47,XXY), and 140 patients showed only 47,XXY. Small testes were observed in 95% of patients and gynaecomastia was seen in 12.4%. Half of the patients showed hypergonadotrophic hypogonadism, while others showed normogonadism (usually hypergonadotrophic). Spermatozoa were observed in semen from one patient with mosaicism and one without. Three-colour FISH revealed hyperploidy in 2.7% and 2.3% of these spermatozoa respectively. Multiple-site testicular biopsies in five recent patients were performed and yielded a specimen with round and elongated spermatids in one patient with 47,XXY karyotype. This sample was cryopreserved for future intracytoplasmic sperm injection. At follow-up, 46% of couples had chosen artificial insemination with donor sperm, and none had chosen adoption. Two patients developed testicular tumours, one a mature teratoma and the other a Leydig cell tumour. Two patients required androgen replacement therapy.     

Zygote intra-Fallopian transfer as an alternative treatment for male infertility

The results of 42 zygote intra-Fallopian transfer cycles in37 couples with persistent male infertility are reported. Of396 retrieved oocytes, 39.1% fertilized normally and 63 zygoteswere replaced in 25 cycles. Twelve patients became pregnant,including two twin pregnancies. The pregnancy rate was 28.6%per started cycle and 48.0% per replacement. The implantationrate per replaced zygote was 22.2% (14 of 63 replaced zygotesimplanted). Sixty-two supernumerary zygotes or embryos werecryopreserved for later use.     

Compound genetic factors as a cause of male infertility: case report

A 40 year old healthy Chinese male with primary infertility was seen in a university male infertility and genetic counselling clinic. He presented with congenital bilateral absence of the vas deferens (CBAVD) and the finding of testis atrophy. Fine needle aspiration mapping of the testis identified and localized sperm production within the testicles for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Careful evaluation of testicular cytology revealed late maturation arrest of spermatogenesis. Cystic fibrosis gene mutation analysis revealed heterozygosity for the 5T variant within the polypyrimidine tract of intron 8. Cytogenetic analysis revealed a pericentric inversion of chromosome 6 with break points at p12 and q21 [46,XY,inv(6)(p12q21)]. This case illustrates that spermatogenesis is not necessarily normal with congenital bilateral absence of the vas deferens. Compound genetic defects may coexist and underlie male infertility.     

Studies on direct intraperitoneal insemination in the management of male factor, cervical factor, unexplained and immunological infertility.

One-hundred-and-twenty couples with male factor, cervical factor, unexplained or immunological infertility underwent direct intraperitoneal insemination (DIPI) of capacitated spermatozoa, combined with ovarian stimulation for a total of 254 cycles. Pregnancy occurred in 47 cycles, resulting in an overall pregnancy rate of 40.5% per patient (two pregnancies occurred in two patients) and a cycle fecundity of 18.5%. Of the patients, 23 have delivered live infants (one twins, 22 singletons), 15 (32%) miscarried and 9 have ongoing pregnancies. The cycle fecundity rates increased with increasing sperm concentration and total progressive motile count, both before and after sperm preparation (r = 0.8161, r = 0.8717 and r = 0.759, r = 0.795, respectively). However, there were two pregnancies where the inseminates had a count of 0.1 x 10(6) total motile spermatozoa. DIPI as an outpatient, in-vivo conception procedure offers encouraging results and is recommended before attempting more costly and invasive assisted reproduction techniques.