1. What is your opinion about the rising male infertility globally?

Response: It is tempting to suggest that the male infertility is rising globally. Worldwide, an estimated 9% of couples meet the definition of infertility, with 50-60% of them seeking care. Taking into account a global perspective, these figures indicate that approximately 140 million people at reproductive age are unintentionally childless or have undergone treatment to procreate. With regard to the male population, it has been estimated that 8% of men within reproductive age seek medical assistance for infertility-related problems, and 1-10% of those have a condition that affects their reproductive potential; varicocele alone is responsible for about 35% of these cases.

The hypothesis that endocrine disruptors and other environmental pollutants, such as insecticides and pesticides, are responsible for declining overall sperm quality have attracted supporters around the world. In fact, taking into account the reports from the International Committee for Monitoring Assisted Reproductive Technology (ICMART), an international non-profit organization that collects data on ART and monitors approximately 2/3 of ART treatments performed worldwide, the numbers of treatments has increased since its first report in 1998, and intracytoplasmic sperm injection, which is mainly intended to bypass severe male factor infertility conditions, has become the most used ART modality.

It is also tempting to suggest that the lower reference limits of semen parameters, as proposed by the recent 2010 WHO manual, are part of gradual declines in sperm count extensively reported over the past two decades. However, there are two other possible explanations that may explain the difference in the reference values between the current and previous WHO manuals. The first is the adherence by many laboratories to higher quality control standards, especially when assessing sperm morphology. The second reason is that previous WHO reference values were obtained mainly based on the clinical experience of investigators who have studied populations of healthy fertile men of unknown “time-to-pregnancy” interval rather than controlled populations of fertile men as in the current edition. With regard to the newly released WHO manual for sperm analysis, everyone must exercise caution when concluding that the newly proposed lowered WHO reference values can be justified by the suggested decline in global sperm quality. It is more probable that such differences are instead related to a methodological bias created by different ways of generating reference values.

  1. If it is true, do you think there is apt number of clinical experts and expertise to tackle this issue?

Response: Despite of not having official estimates, I do believe there is a lack of male fertility experts able to take care of the increasing numbers of infertile men seeking care. Urologists are the primary experts to assess male infertility patients. They are trained to diagnose, counsel, provide medical or surgical treatment whenever possible and correctly refer the male patient for assisted conception. By integrating the reproductive team, the urologist may be responsible for the above-cited tasks and to perform surgical sperm retrieval. However, I feel that most urologists are not very enthusiastic about treating male infertility patients as they are with other urological conditions. In general, they are very busy performing other urological tasks, and in several occasions I was dismayed to see urologists who integrate assisted reproductive teams acting as mere “technicians”, i.e., coming to the field to perform sperm retrievals rather than providing a comprehensive male evaluation and discussing the therapeutic options with the infertile couple.

Urologists willing to act as male infertility experts should be aware of the advances in the field of male reproductive medicine and embrace the cause. I feel that male infertility specialists are in great demand and young urologists should be aware of the opportunities that the fields of andrology and male reproduction have to offer.

  1. Do you think Andrology is still lagging behind in terms of clinics offering the right options for patients?

Response: I do believe so. Several IVF Clinics are run by either REI specialists or gynecologists and little attention is paid to the infertile men. I am not aware of the worldwide situation but can give a testimonial of the current situation in our country. In Brazil, there is more than 160 IVF Clinics and the vast majority does not have a consulting urologist integrating their staffing. As I often hear in meetings and informal discussions with my GYN and REI peers, I quote them: “…as soon as the man can provide a semen sample to be used for IUI, IVF or ICSI, it is fine”.

Andrology, the branch of medicine concerned with male reproductive health, emerged as a specialty at the end of the nineteenth century. However, it was not until the latter half of the past century that andrology had been recognized as one of the most intriguing sub-specialties of human reproduction. This is due to rapidly rising volume of scientific evidence that documents the critical role of spermatozoa in the fertilization process. Due to the true nature of research involving the classical disciplines of physiology, biochemistry and molecular biology, scientific knowledge and application of andrology evolve continuously. Invariably, it is leading to a growth in andrological information and better understanding of the field of male reproductive biology.

The scope of modern andrology now covers a wide spectrum from genetic studies to pubertal changes in the male and from infertility and assisted reproduction techniques to disorders of the prostate, sexual function and contraception. As such, extraordinary advances have been achieved in the field of male infertility in the last decades. There are new concepts in sperm physiology and several modern tools for the assessment of spermatogenesis kinetics in vivo. New tests using molecular biology and DNA damage assays allow the clinician to correctly diagnose men so far classified as having idiopathic male infertility. Most importantly, infertility clinics and doctors should not limit couple’s options for treatment based on their own technical limitations, but always provide all treatment options available for that particular case scenario. As such, couples should be properly counseled of all treatment modalities available in relation with their case, in order to be able to make an unbiased decision and selecting the most appropriate option fitting their needs.

  1. If oligozoospermic or Azoospemic, straight away ICSI!! The suggestion of many clinics. Isnt there any alternatives?

Response: Of course there are other options. As an example, in a group of 2,875 infertile couples who attended our tertiary center for male reproduction we identified conditions that could potentially be corrected with surgical procedures in approximately one third of the male partners. In the field of treatment, microsurgery has increased success rates either for reconstruction of the reproductive tract or the retrieval of spermatozoa for assisted conception.

Varicocele treatment based on the presence of clinically palpable varicocele and abnormal semen parameters should be considered as a treatment option for infertile males. Open microsurgical inguinal or subinguinal techniques are currently the best treatment modalities for varicocelectomy because they result in higher spontaneous pregnancy rates and fewer recurrences and postoperative complications than laparoscopic, radiologic embolization and macroscopic inguinal or retroperitoneal varicocelectomy techniques. Although there are no absolute predictive factors for successful varicocele repair, surgical repair of varicocele may improve semen parameters and functional markers of oxidative stress and DNA integrity. The chance for either spontaneous or

assisted conception is increased after the repair of clinical varicocele. In one meta-analytic study by Marmar et al. (2007), the authors showed that the chances of obtaining a spontaneous conception were 2.8 times higher in the varicocelectomy group as compared to the group of patients who received either no treatment or medication. New studies have also shown that men who are treated of their varicocele before assisted conception may benefit as well. Our group has recently demonstrated (Esteves et al, 2010) that the treatment of clinical varicoceles may improve the outcome of ICSI in couples with varicocele-related infertility. In our study, the chances of live birth were significantly increased by 1.9-fold, and the chance of miscarriage was reduced by 2.3-fold if the varicocele had been treated before assisted conception. In addition, a recent meta-analysis (Weedin et al. 2010) reported appearance of sperm in ejaculates of 39% of azoospermic individuals whose varicoceles had been treated. In their study, testicular histopathology results were predictive of success. Postoperatively appearance of sperm in the ejaculates was increased 9.4-fold in patients with biopsy-proven hypospermatogenesis (HS) or maturation arrest (MA) than in Sertoli-cell only. Although the use of motile ejaculated sperm is preferred for ICS, persistent azoospermia after varicocele repair is still a potential problem and sperm extraction before ICSI will be inevitable for many individuals. In these circumstances, successful sperm retrieval rates of 60% have been reported using testicular microdissection (micro-TESE) sperm extraction. It is suggested that varicocele repair may maximize the chances of retrieving sperm for ICSI in azoospermic men with clinical varicoceles by 2.6-fold.

Men with obstructive azoospermia may father children either by surgical correction of the obstruction, which may allow the couple to conceive naturally, or retrieval of sperm directly from the epididymis or testis, followed by ICSI. Sperm return to ejaculates after microsurgical reconstructions are achieved in 70% to 95% of cases, and 30% to 75% of couples achieve unassisted pregnancy. Patency and pregnancy after microsurgical vasectomy reversal are inversely related to the interval of obstruction since vasectomy. Other factors that affect success rates include the intraoperative appearance of vasal fluid, the presence or absence of sperm in the vasal fluid and their quality, the length of the remaining segment adjacent to the epididymis, the age of the female partner, and the experience of the surgeon.

Transrectal resection of the ejaculatory duct is the treatment of choice for ejaculatory duct obstruction. After TURED, sperm return to the ejaculate in approximately 50% to 75% of men, and approximately 20% of couples achieve pregnancy.

Lastly, emerging evidence suggests that life-style and environmental conditions are of utmost importance in male fertility and subfertility; there are treatment options for infertile men fitting in these categories that may enhance their odds of achieving spontaneous or assisted conception.

  1. What is your suggestion to patients who are going for ICSI? Like

  1. Should they undergo any genetic testing?

R: Male infertility can be associated with different genetic factors that include chromosomal aberrations, genetic alterations and Y-chromosome microdeletions. Chromosomal aberrations are assessed through G-band karyotyping. Genetic mutations and Y-chromosome microdeletions assessments are also performed by analysis of peripheric blood sampling. DNA is amplified using polymerase chain reaction (PCR) biomolecular technique. Chromosomal abnormalities may be present in approximately 6% of infertile men and its prevalence is inversely correlated with sperm count. Azoospermic men can be affected in as much as 16% of cases. Sex chromosomal aneuploidy (Klinefelter syndrome; 47,XXY) is the most frequent chromosomal disorder present in infertile men and is generally associated with hypotrophic or atrophic testicles, elevated serum FSH levels and azoospermia, although spermatogenesis can be differently affected in patients with a mosaic karyotype (46,XY/47,XXY). Among genetic disorders, the mutation of the cystic fibrosis gene (CFTR gene) located on the long arm of chromosome 7 is the most commonly found. According to the extension of the mutation, cystic fibrosis can be manifested at its full clinical presentation (an autosomic recessive potentially fatal disease) or in a mild form, where congenital bilateral absence of the vasa deferentia exists and affects approximately 1.3% of infertile men. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations compromise the development of Wolffian ducts-derived structures (efferent ducts, epididymis and vasa deferentia) and may also implicate in seminal vesicles hypoplasia or agenesis and unilateral renal agenesis. Approximately 80% of men presenting with CBVAD have a CFTR mutation and as the genetic testing is not 100% sensitive, these subject should be assumed to harbor the genetic anomaly. Testing should be offered to his female partner as she may also be a carrier (approximately 4% risk) before using his sperm for assisted conception. After genetic testing, genetic counseling should be offered. Recent data suggest that azoospermic men with idiopathic obstruction and those presenting the triad composed by chronic sinusitis, bronchiectasis and obstructive azoospermia (Young Syndrome) have an elevated risk for CFTR mutation. The long and short arms of Y-chromosome are respectively related to spermatogenesis and testicle development. The Y-chromosome region related to infertility is called azoospermia factor locus (AZF – azoospermia factor). The locus can present complete or partial microscopic deletions, isolated or in combination, and in non-overlapping subregions called AZFa, AZFb, AZFc and AZFd (Figure 3). These subregions contain multiple genes controlling different steps of spermatogenesis. The most common Y-chromosome deletion in infertile men is the one affecting the DAZ gene (deleted in azoospermia) located in AZFc region. Severe oligozoospermia or azoospermia can be seen in such cases. Y-chromosome microdeletions are found in 15% of men with azoospermia and less often in 6% of men presenting with severe oligozoospermia (<1 million/mL). For sperm counts between 1 and 5 million/mL the detection rate drops down to 1.7%. Detection of Y-chromosomal microdeletions provides predictive information on the success of sperm retrieval for intracytoplasmic sperm injection (ICSI). AZFa and AZFb microdeletions present as azoospermia and are associated with germ cell aplasia and maturation arrest, respectively. In such cases, sperm retrieval attempt is not recommended because there is virtually no chance of finding testicular sperm.

In AZFc microdeletion cases sperm can be retrieved in approximately 71% of patients. Clinical pregnancy rates are virtually the same as for idiopathic azoospermic patients who can have their sperm retrieved as well.

Testing also yields information for genetic counseling as sons of men with Y-chromosomal microdeletion will inevitably inherit the abnormality and may also be infertile. Structural abnormalities in the autosomes, such as inversions and translocations, are also higher in infertile men. Gross karyotypic abnormalities are related to an elevated risk for miscarriages and for having children with both chromosomal and congenital defects. As such, men with nonobstructive azoospermia or severe oligozoospermia should be karyotyped before their sperm are used for ICSI.

  1. Any lifestyle modifications for 3-6 months prior to ICSI cycle to get the robust sperm?

R: Absolutely. For example, cigarette smoke generates high levels of oxidative stress, directly increasing seminal leukocyte concentrations and seminal ROS generation, and decreasing seminal levels of the antioxidant enzyme superoxide dismutase. Smoking decreases concentrations of the seminal plasma antioxidants thereby reducing the oxidant scavenging capacity of the spermatozoa and seminal fluid. Moreover, studies show that live birth delivery is reduced by 3.7-fold in couples undergoing IVF who smoke. We should advise our patients to stop smoking for at least 2 months before ART, and this time lag is in accordance to the novel concept of the spermatogenic cycle duration of approximately 64 days.

Regarding alcohol, few studies suggest that live birth delivery is reduced by 5.5-fold and miscarriage increased by 2.7-fold in heavy drinkers undergoing IVF. The effect of stress is still debatable and caffeine consumption seems to be not detrimental. Although morbid obesity is associated with unfavorable IVF/ICSI cycle outcome, as evidenced by lower pregnancy rates in females, there is no evidence for a contributing male factor when assisted reproductive methods are used.

I also recommend to increase the consumption of antioxidant-rich food: carrots, spinach, tomatoes, papaya, guava, cherries, melons and peaches are rich in β-carotene; guava, kiwi, mango, pineapple, melons, strawberries, berries, tomatoes, broccoli, cabbage, oranges, lemons and other citrus fruits are rich in vitamin C; lettuce, peanuts, almonds, coconut, corn, soy or olive oil; wheat and corn germ are rich in vitamin E; asparagus, potatoes, vegetables, eggs and fish are rich in zinc.

  1. Should antioxidants for 3-6 months help these patients to reduce ROS and DNA fragmentation?

R: Considering that modern lifestyle is characterized by a relatively low intake of natural antioxidants like vegetables or fruit, and a relatively high exposure to exogenous sources of ROS such as alcohol and cigarette consumption or environmental pollutants, and considering that infertile patients that we routinely see in our practice are at risk of excessive Oxidative Stress, there is a rationale to support the use of antioxidant treatment for at least 2 months before ART.

Reactive oxygen species are products of aerobic metabolism. At certain levels they are not detrimental, but several conditions induce an overproduction of ROS, causing oxidative stress. Oxidative stress is higher in infertile men than fertile ones. Regardless of its cause, the end product of OS is the oxidation of sperm structures that become dysfunctional. The impairment of sperm function caused by OS may be either the only factor causing infertility or a contributor to a recognized disease that cause infertility. Options to minimize OS include: i) the treatment of the underlying pathology, if possible, ii) the removal of risk factors, which is not always feasible, or iii) by the administration of antioxidant supplementation. A recent Cochrane review suggested that antioxidants use improved the chances of pregnancy for subfertile men undergoing assisted conception. I routinely prescribe a combination of vitamic C 500mg, vitamin E 400 UI, folic acid 2 mg, zinc 25 mg, and selenium 26 mcg.

  1. Any microsurgeries?

R: As already mentioned, it has been shown that microsurgery may also improve the outcomes of assisted reproduction in couples with varicocele-related infertility. Esteves et al. (2010) observed higher pregnancy rates after ICSI in the group of men who underwent microsurgical varicocele repair before ART (60.3% versus 45.0%), and logistic regression showed that the chance of obtaining a clinical pregnancy was increased by 69% if the varicocele had been treated before ICSI (OR: 1.69, 95% CI 1.00–2.84). Also, the chance of having a miscarriage after ICSI was significantly reduced by 2.3 times if the varicocele had been treated (OR: 0.433; 95% CI 0.22-0.83; P=0.01). Inci et al. (2009) reported that treatment of clinical varicoceles prior to SR increased the chance of obtaining testicular sperm using micro-TESE in a group of NOA individuals with clinical varicoceles (54). Retrieval rates were 53% and 30% in the treated and untreated men, respectively (odds-ratio [OR]: 2.63; 95% confidence interval [CI] of 1.05–6.60). In both conditions, it is my approach to discuss microsurgery for varicocele treatment with my patients before embarking in IVF-ICSI. Moreover, microsurgery is my primary option to extract sperm in men with nonobstructive azoospermia. Microdissection testicular sperm extraction (micro-TESE) has been shown to be more successful in sperm retrieval than a single biopsy or multiple random biopsies. In addition, micro-TESE seems to have less effect on testicular function because it spares vessels during dissection and removes less tissue than random biopsies.

  1. Any antibiotics?

Response: Subclinical genital infections are not uncommon in the infertile male population. In non-selected cases, the prevalence of asymptomatic male genital tract infection varies between 10 and 20% and amounts to up to 35% in a large study comprising of more than 4000 patients consulting for infertility. Bacterial growth in seminal cultures is often halted by the anti-bacterial properties of zinc, which is an integral element of seminal plasma. Similarly, it is difficult to grow Chlamydia and Micoplasma in culture. Moreover, seminal leukocytes, even at low levels, generate 1000x more ROS than defective spermatozoa. For these reasons, I routinely prescribe an one-dose (1000mg) of azithromycin to both the man and his spouse before assisted conception. such a practical approach is to simply prescribe broad-spectrum antibiotics against gram-negative bacteria commonly found in the semen.

  1. Some lab people from their experience mention that “ plenty of bacteria in semen (high power field) and when they sent for culture “no growth”. Are these bugs anaerobic not showing in the culture plates? Moreover embryologists from tropical countries suggest presence of bacteria in testicular tubules. So are these bugs are making its way retrograde from accessory glands to testicular milieu where they can evade immune reactions and living in a symbiotic nature.

Response: Most of these bacteria come from the skin and are often gram-positive Staphylococcus epidermidis. S. epidermidis are part of the skin flora and are not usually pathogenic. The best way to get rid of them is to give proper instructions to the patient to wash the hands and penis before masturbation for sperm collection purposes. Also, I perform a through skin cleaning before sperm retrieval. As already mentioned, the anti-bacterial properties of zinc prevent most bacteria to grow in culture.

  1. For IUI/IVF do you suggest telling patient frequent ejaculations (wash effect) prior to their actual cycles

Response: Yes, I do recommend frequent ejaculation. Moreover, it has recently been shown that short abstinence time decrease the frequency of sperm with fragmented DNA in ejaculates.

  1. Does sperm quality really affect embryo ad blastocyst rate?

Response: Spermatozoa are highly specialized cells with the purpose of not only delivering competent paternal DNA to the oocyte but also to provide a robust epigenetic contribution to embryogenesis. Currently, sperm indexes determination in the clinical setting is generally based on cell morphology and DNA content. Both sperm morphology and DNA integrity results, obtained from raw semen samples, have been shown to be of prognostic value for unassisted and assisted conception and useful in the selection of the best assisted conception modality.

These assays, however, provide an assessment of the distribution of cells in a given ejaculate that may not be representative of the sperm population used in the ART treatment cycle. In fact, severe teratozoospermia, using Kruger’s strict criteria on pre-ART semen analysis, does not correlate to fertilization and embryo formation (including blastocyst development) in ICSI cycles. Nonetheless, if a more holistic approach to sperm morphology is taken, two prognostic groups can still be identified in cases of severe teratozoospermia (<4% normal) because certain morphology patterns and sperm abnormalities are known to affect ICSI outcomes. The first group includes mostly genetically determined sperm pattern defects, such as globozoospermia, short tail syndrome and small-headed spermatozoa (in most cases combined with very small acrosomes). All of these types represent untreatable conditions that have been associated with abnormal sperm function and poor ART outcomes. The second group includes unspecified or non-genetically determined sperm defects or patterns caused by environmental factors, medication, infection and related infertility conditions, including varicocele. Treatment of these conditions has been shown to optimize sperm morphology indexes with a positive impact on ART outcomes. Although the embryologist microscopically selects morphologically normal individual sperm during ICSI, form normalcy does not necessarily imply normal DNA content. As such, sperm DNA testing has been advocated to be an independent and reliable marker of fertility potential since sperm chromatin and DNA integrity is essential to ensure that the fertilizing sperm can support normal embryonic development of the zygote. At present, conflicting reports exist on the role of sperm DNA fragmentation index for embryo development, and it is apparent that DNA fragmentation does not significantly impair zygote and cleaving embryo morphology because major activation of the embryonic genome only begin after the 4-cell stage. These observations do no underscore the importance of finding ways to increase sperm DNA integrity, since it has been suggested that DNA fragmentation is associated with late paternal effects that may lead to early miscarriages or diseases in the offspring. The etiology of sperm DNA damage is multi-factorial and may be due to primary (ageing, cryptorchidism, genetic defects, idiopathic) and or secondary (drugs, environmental, tobacco smoking, genital tract inflammation, infection, testicular hyperthermia and varicoceles) factors. Specific or non-specific treatments, including antioxidant supplements, are generally associated with reduced levels of sperm DNA damage and/or improved fertility potential.

Taken in conjunction, it is apparent that there is no unique sperm factor able to predict embryo development, but several candidate biomarkers are involved in this complex process. As a result, a wide variety of techniques have been proposed, including externalization of phosphotidylserine (magnetic-activated cell sorting), cell charge (zeta charge), maturity markers (hyaluronic acid binding) and detailed morphological analysis (intracytoplasmic morphologically selection sperm injection). Currently, these are several shortcomings for the routine application of these new methods to a busy IVF laboratory, both financially and logistically, and current data fail to indicate superiority of any of these methods over conventional ICSI. It is clear that better sperm fertility tools are urgently required. In this context, metabolomics and proteomic sperm profiling are under investigation and may be translated into clinical practice in the near future.

  1. IS testicular sperm better than ejaculated sperms? If the patient has few ejaculated sperms do you suggest to go for TESE

Response: Recently, levels of DNA damage in ejaculated and testicular spermatozoa collected on the same day of the intracytoplasmic sperm injection (ICSI) procedure have been compared. Results indicate that ejaculated spermatozoa show three-fold higher DNA damage when compared with testicular samples in men with persistent high DNA damage on ejaculated sperm. Moreover, there is an increased risk of miscarriage in pregnancies obtained by ICSI in couples whose male partners have high levels of DNA damage. It has also been shown that reproductive outcomes are better by using testicular sperm rather than ejaculated sperm in couples undergoing failed ICSI attempts. As such, I recommend the use of testicular sperm rather than ejaculated sperm for ICSI in cases of persistent high DNA damage. In cases of patients with very few ejaculated spermatozoa I do suggest to perform ICSI by splitting the oocytes and performing half of sperm injections with ejaculated sperm and the other half with surgically-extracted testicular sperm (either by TESE or TESA).

  1. Do you believe all ICSI or do according to the semen quality!!!

Response: In terms of ART, I advocate using the results of sperm morphology by strict criteria, number of motile sperm post-washing and DNA fragmentation testing to select the most suitable treatment modality. In our hands it is either IUI or ICSI. Based on the published literature and on our 15-year experience with ART, I do not believe conventional IVF offers any significant advantage compared to ICSI. Conversely, follow-up studies are reassuring with regard to the health of offspring derived from ICSI as compared to IVF. In general, IVF techniques are associated with multiple gestation and an increased risk of congenital abnormalities (including hypospadias). ICSI in particular may carry an increased risk of endocrine abnormalities and

epigenetic imprinting effects, but the absolute risk of any of these conditions remains extremely low. However, current data is limited and studied populations are heterogenic. Therefore, conceived children should be closely monitored. I acknowledge that costs and reimbursement issues may play significant roles regionally, but at present my opinion is that the technical debate of using standard IVF or ICSI is trivial.

A list of recent publications by the author is provided below, and can be downloaded from his website at: http://www.androfert.com.br/site/pasta_124_0__artigos-publicados-em-periodicos.html

1. Esteves SC, Hamada A, Kondray V, Pitchika A, Agarwal A. What every gynecologist should know about male infertility: an update. Arch Gynecol Obstet 2012; DOI 10.1007/s00404-012-2274-x

2. Miyaoka R, Esteves SC . A critical appraisal on the role of varicocele in male infertility. Adv Urol 2012; 1:1-9.

3. Esteves SC, Zini A, Aziz N, Alvarez JG, Sabanegh ES, Agarwal A. Critical appraisal of World Health Organization's new reference values for human semen characteristics and effect on diagnosis and treatment of subfertile en. Urology 2012; 79:16-22.

4. Esteves SC, Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.

5. Esteves SC. Chromosomal and molecular abnormalities in a group of Brazilian infertile men with severe oligozoospermia or non-obstructive azoospermia attending an infertility service [Editorial comment]. Int Braz J Urol 2011; 37:244-251.

6. Esteves SC, Miyaoka R, Agarwal A . An update on the clinical assessment of the infertile male. Clinics 2011; 66:691-700.

7. Esteves SC, Miyaoka R, Agarwal A . Sperm retrieval techniques for assisted reproduction. Int Braz J Urol 2011; 37: 570-583.

8. Hamada A, Esteves SC, Agarwal A. Unexplained male infertility. Hum Androl 2011; 1:2-16.

9. Esteves SC, Miyaoka R, Agarwal A . Surgical treatment of male infertility in the era of intracytoplasmic sperm injection - new insights. Clinics 2011; 66:1463-1478.

10. Agarwal A, Esteves SC. Advances in andrology and male reproductive health [Editorial]. Open Reprod Sci J 2011; 3: 1-6.

11. Agarwal A, Esteves SC. Impact of the new WHO guidelines on diagnosis and practice of male infertility. Open Reprod Sci J 2011; 3: 7-15.

12. Esteves SC, Agarwal A. What is new in the clinical assessment and tTreatment of the infertile male. Open Reprod Sci J 2011; 3:16-26.

13. Hamada A, Esteves SC, Agarwal A. The role of contemporary andrology in unraveling the mystery of unexplained male infertility. Open Reprod Sci J 2011; 3:27-41.

14. Dada Rima, Esteves SC, Agarwal A . Genetic testing in male infertility. Open Reprod Sci J 2011; 3:42-56.

15. Esteves SC, Verza Jr S. Relationship of in vitro acrosome reaction to sperm function: an update. Open Reprod Sci J 2011; 3:72-84.

16. Esteves SC, Schneider DT. Male infertility and assisted reproductive technology: lessons from the IVF. Open Reprod Sci J 2011; 3:138-153.

17. Esteves SC. - Varicocele repair in patients with nonobstructive azoospermia: a meta-analysis [Editorial comment]. J Urol 2010; 183:2315.

18. Esteves SC, Oliveira FV, Bertolla RP. Clinical outcome of intracytoplasmic sperm injection in infertileen with treated and ntreated clinical aricocele. J Urol 2010; 184:1442-1446.

19. Verza Jr S, Feijo CM, Esteves SC. Resistance of human spermatozoa to cryoinjury in repeated cycles of thaw-refreezing. Int Braz J Urol 2009; 35:581-591.

20. Esteves SC, Schertz JC, Verza Jr S, Schneider DT, Zabaglia SFC. A comparison of menotropin, highly-purified menotropin and follitropin alfa in cycles of intracytoplasmic sperm injection. Reprod Biol Endocrinol 2009; 7:111.

21. Esteves SC. Sperm retrieval and intracytoplasmic sperm injection in men with nonobstructive azoospermia, and treated and untreated varicocele [Editorial comment]. J Urol 2009; 182:1504-1505.

22. Verza Jr S, Esteves SC. Sperm defect severity rather than sperm source is associated with lower fertilization rates after intracytoplasmic sperm injection. Int Braz J Urol 2008; 34:49-56.

23. Esteves SC, Schneider DT, Verza Jr S. Influence of antisperm antibodies in the semen on intracytoplasmic sperm injection outcome. Int Braz J Urol 2007; 33:795-892.

24. Esteves SC, Sharma RK, Thomas Jr AJ, Agarwal A. Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test. Int Braz J Urol 2007; 33:364-376.

25. Esteves SC, Spaine DM, Cedenho AP. Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method. Braz J Med Biol Res 2007; 40:985-992.

26. Esteves SC, Glina S. Recovery of spermatogenesis after microsurgical subinguinal varicocele repair in azoospermic men based on testicular histology. Int Braz J Urol 2005; 31:541-548.