015PG Vasectomy Reversal and Male Infertility Treatment in the ICSI Era

Home , Vasectomy reversal, Vasoepididymostomy, Vasovasostomy

Saturday, May 16, 2015 8:30 AM – 11:30 AM

Faculty

Peter N. Schlegel, MD - Course Director Sheldon H.F. Marks, MD Robert D. Oates, MD

Vasectomy Reversal and Male Infertility Treatment in the ICSI Era

Peter N. Schlegel, MD, Course Director, Professor & C hairm an, W Urologist-in-Chief, New York-Presbyterian/Weill Cornell, N e w Y o rk , N Y

Sheldon H.F. Marks, MD Director, International Center for Vasectomy Reversal, PC, Tucson, AZ

Robert D. Oates, MD Professor of Urology, Vice-Chair, Department of Urology Boston University School of Medicine B o s to n , M A

Disclosures: Dr. Schlegel is a medical advisory board consultant to Theralogix, Inc. Drs. Marks and Oates have no disclosures at this time (updated information available at the AUA meeting.)

Schedule:

8:30-8:35 Introduction Dr. Schlegel

8:35-9:20 Evaluation of azoospermic male Dr. Oates

9:20-9:30 Questions & Answers

9:30-10:15 Vasovasostomy & Vasoepididymostomy Dr. Marks

10:15-10:30 Questions & Answers

10:30-10:45 Break

10:45-11:05 Sperm retrieval techniques Dr. Schlegel

11:05-11:15 Evolving sperm genetic evaluation Dr. Schlegel

11:15-11:30 Cases, Questions & Answers All faculty

The etiology and differential diagnosis of azoospermia will be discussed, emphasizing the differentiation of obstructive from non-obstructive causes. Microsurgical methods of vasovasostomy and vasoepididymostomy will be shown, including practical tips and video presentation with expected results according to the AUA Best Practice Policy committee recommendations.

Objectives: After attending this course, participants should be able to:

• describe the differential diagnosis of azoospermia, and the techniques and results of various methods of sperm retrieval from azoospermic men for IVF and ICSI • understand the importance of proper genetic testing before reconstructive or sperm retrieval procedures in azoospermic men • Identify novel tests that can be used to identify male factors for couples who have failed ICSI treatment • describe the surgical approaches, as well as the preoperative and intraoperative factors, that influence the results of vasovasostomy and vasoepididymostomy • discuss the effect of genetic abnormalities on the ability to retrieve sperm from men with nonobstructive azoospermia and the transmission of genetic abnormalities to offspring produced by sperm retrieval with IVF and ICSI

1 Evaluation of the azoospermic male Robert D. Oates, M.D.

Evaluation of the azoospermic male: Azoospermia is defined as the absence of sperm in the ejaculate. It is imperative to examine at least two specimens before concluding the male is azoospermic. It is not unusual to have tiny numbers of sperm seen in the pellet after centrifugation that must be a part of the semen analysis protocol for azoospermic samples. In evaluating the azoospermic male, it is helpful to look at semen volume and pH, which allows a differentiation into low volume and normal volume azoospermia. The seminal vesicles contribute approximately 70% of the fluid to the normal ejaculate, which is alkaline (pH 8.0), and contains fructose. The vasa deliver approximately 10% and the acidic (pH 6.5) prostatic fluid component the remaining 20%. The seminal vesicle and vasal fluids are delivered to the prostatic urethra via the paired ejaculatory ducts. Therefore, if the seminal volume is normal (2.0cc) and the pH is alkaline (8.0), the seminal vesicles must be present and functional and the ejaculatory ducts must be patent. There is no need for a fructose assay since the volume and pH alone indicate the presence of seminal vesicle fluid and patency of the ejaculatory ducts. In normal volume azoospermia, the differential diagnosis does not, therefore, include Bilateral Ejaculatory Duct Obstruction (EDO) or Congenital Bilateral Absence of the Vas Deferens (CBAVD; vide infra). In both EDO and CBAVD, the semen volume is low and the pH is acidic as the seminal vesicles are normal but blocked in EDO and absent or atretic in CBAVD. However, the differential does include spermatogenic compromise or an obstructive process higher up than the ejaculatory ducts (most often in the scrotum at the level of the vas (e.g. prior vasectomy) or epididymis (prior inflammation). Prior history, physical examination, and hormonal assays can typically clarify these two possibilities in normal volume azoospermia. Physical examination cannot be overemphasized. In low volume, low pH azoospermia, there is no contribution to the ejaculate from seminal vesicle fluid and only prostatic fluid is present. No fructose assay is necessary as there is no seminal vesicle fluid present and the assay will be negative. The differential includes EDO and CBAVD. Physical examination will make the diagnosis in an instant – In EDO the vasa are present, palpable, and may feel “full” secondary to more distal obstruction. The testes will be normal size and consistency. In CBAVD, the vasa will not be palpable, there will always be at least a caput epididymis present and the testes will be of normal size and consistency. Sperm aspiration is almost always successful and the sperm can be used for ICSI. A preliminary differential diagnosis can be formulated just based on knowledge of the semen volume and pH. History and physical examination then direct the work-up from that point. It is also necessary to determine whether the azoospermia is secondary to blockage of sperm produced by normal testes (obstructive azoospermia; OA) as may happen in EDO, CBAVD, a post- inflammatory epididymal blockage, etc, or whether the azoospermia is due to inadequate or absent spermatogenesis with a normal ductal system (non-obstructive azoospermia; NOA). In OA, the testes will be normal size and consistency as spermatogenesis is adequate (the bulk of the testis mass is comprised of the seminiferous tubules). No testis biopsy is necessary as the histological pattern will only confirm what is already suspected – spermatogenesis is not the problem. Conversely, in NOA, the testes are typically smaller and softer than normal as spermatogenesis is compromised and the total mass of the seminiferous tubules is reduced. The epididymis and vasa are normal to palpation. No testis biopsy is necessary as the histological pattern will only confirm what is already suspected – spermatogenesis is the problem. History may also direct one to the diagnosis: prior chemotherapy would suggest NOA, prior inguinal hernia repair might be a clue to obstruction, etc. Finally, hormonal data will be additive and, possibly, definitive if the history and physical examination are not diagnostic. Follicle stimulating hormone is released by the pituitary and is regulated by a negative feedback loop involving inhibin, the two subunits of which are secreted by Sertoli cells and germ cells. When spermatogenesis is normal, the hypothalamic-pituitary-gonadal axis works without strain and the FSH level will typically be in the lower aspects of the reported reference range. For an assay with a reference range – not to be interpreted as a “normal range” – of 2 – 20, for example, the value in a patient with normal sperm production will be roughly 2-5. When spermatogenesis is compromised, less inhibin is secreted, there is less negative feedback on the hypothalamus/pituitary and the output of FSH rises. The old adage that the FSH must be “2-3 times the upper limit of normal” to be indicative of spermatogenic failure is not true. As the FSH steadily climbs out of the lowest aspects of whatever that particular assay’s reference range, the more likely the spermatogenic process is flawed and dysfunctional. In a patient with normal volume azoospermia, slightly small and soft testes and palpably normal ductal structures, even an FSH of 7 or 8 suggests compensatory output due to spermatogenic compromise and, therefore, NOA. Finally, the question of whether an azoospermic male is taking anabolic steroids / testosterone supplementation is a critical one. Men are being prescribed testosterone (gels or injections) in increasing numbers and the body building culture is growing as well (they may be taking a variety of anabolic agents). It is necessary for testosterone to be produced within the testis, otherwise spermatogenesis will be disrupted. If testosterone (or any anabolic) is coming from an exogenous source, the pituitary does 2 not need to release LH (or FSH as well) and, as a result, the Leydig cells within the testis will not be stimulated to produce testosterone (as far as the body knows, there is plenty around) and, consequently spermatogenesis will be reduced/stopped. In an azoospermic male in whom you suspect this to be occurring (and who does not admit to taking anabolics), his testosterone values will be high while his LH and FSH will be very suppressed. The treatment is to have the patient stop the testosterone/anabolic use and let his own system restore itself which it typically will do over a week or so (the hypothalamic pituitary axis) and a few months later there will be sperm in the ejaculate (quite variable, however). Measurement of Estradiol is important in these cases as well since not all of the anabolic compounds taken are easily measured by the assays we have. In the case where a man is taking one of these preparations, his FSH and LH will be suppressed, his Testosterone will be low or not detectable but his Estradiol (arising from conversion of androgen via aromatase) will be normal or high – proof of anabolic use.

Genetic testing in the azoospermic male: Genetic studies, as described below, are necessary to obtain prior to any surgical intervention in men with NOA (no definable, obvious cause such as prior chemotherapy) or CBAVD. Darwinian evolution speaks about genetic change that allows improved survival and adaptation. The many branches of the evolutionary tree are now being refined with increased genetic knowledge, a supplement to form and function which have been the determinants so far as to where a species was located on that tree. All along, however, reproduction must have been maintained at a high level for any of those changes, those evolutionary modifications, those adaptive alterations in the genome to be passed along to succeeding generations. The reproductive axis has been, therefore, equally as important and equally invested. Couple this supposition with the knowledge that meiosis, an incredibly complex and intricate process, occurs only during spermatogenesis and oogenesis. Common sense certainly suggests, then, that the reproductive machinery may involve many genetic cogs (genes, RNAs, proteins, etc) specific to it and that their dysfunction may not impair the overall health of the individual – just his ability to procreate. This train of thought implies that all idiopathic NOA men have a genetic aberration of some sort – only a small percentage of which we presently understand and can detect. It is appropriate, however, to learn about these genetic etiologies as best we can prior to moving forward with surgical intervention, use of any retrieved sperm, and ICSI. There are two blood tests that are currently available that should be obtained in the NOA patient prior to instituting any therapy. The Y chromosomal microdeletion assay is a PCR based analysis of the Y chromosome used to determine if microdeletions are present or not. Microdeletions are cytogenetically unrecognizable deletions of various stretches of the chromosome that have been mapped out and shown to be of clinical importance. There are two regions of the Y that contain genes involved only in the spermatogenic process and, when these regions are partially or completely missing so are the “spermatogenic” genes that normally reside there and sperm production is disrupted to a variable degree. The first of these areas is termed AZFa (AZF is an acronym for “azoospermia factor”). AZFa is located on the long arm of the Y chromosome and contains two genes of importance, DDX3Y and USP9Y. Microdeletion of the AZFa region is found as the proximate cause of the NOA in approximately 1% of such men. The necessity of discovering this prior to any surgical intervention (TESE) is that available data suggest that no sperm will be found upon TESE - that the spermatogenic defect induced by the absence of the AZFa region is so profound that TESE will not yield sperm, that TESE will not be helpful, and that TESE should not be performed. An AZFa microdeletion is prognostic of TESE failure. The second region that a Y chromosomal microdeletion assay may identify as microdeleted was previously thought to be two distinct regions, AZFb and AZFc. Recent data shows that the AZFb and AZFc regions are simply subregions within one long stretch that includes both and that AZFb and AZFc actually overlap slightly. When an AZFb or AZFb/AZFc microdeletion is identified (also known as P5/ proximal P1 and P5 /distal P1, respectively), there is little or no chance that spermatozoa will be found on TESE. With these findings, occurring in 1-2% of NOA men, TESE is not helpful and, once again, the Y chromosomal microdeletion assay is prognostic of failure. The AZFc region (also known as b2/b4) is microdeleted in 1:2500 men, 13% of NOA men and 6% of severely oligospermic men. In NOA men with an AZFc microdeletion, sperm can be recovered from the testis tissue approximately 60-70% of the time and that sperm can be used in the fresh or frozen- thawed state as a functionally competent source of sperm for ICSI. As can be realized, the spectrum of spermatogenic deficiency in AZFc microdeleted men ranges from complete (no spermatozoa even upon TESE) to severe oligospermia (<5 x 106/cc). This microdeleted Y chromosome will be passed directly on to any conceived male offspring, their spermatogenic profile expected to be the same as described above and not necessarily that of the father’s. That is, the conceived male may be at the most severe end of the spectrum when he attains reproductive age (sterility) even though his father (our patient) had sperm to use for ICSI (either ejaculate or testis). The couple deserves to be informed of this so that they can make active choices for themselves. These include not using any retrievable sperm but using donor sperm, 3 using any retrievable sperm for ICSI and if a male child is born to inform him of his aberrant Y chromosome and its meaning when the time is right, or using any retrievable sperm for ICSI while employing preimplantation genetic diagnosis to avoid the transfer of any male embryos and prevent infertility/sterility from affecting any offspring. The couple can only consider those choices if they are provided the data that a Y chromosomal microdeletion assay may provide. A karyotype visually depicts the structure and the number of the patient’s chromosomes. In the NOA patient group, approximately 10% of men will be found to have the 47,XXY chromosomal constitution of Klinefelter syndrome. There is no classic Klinefelter phenotype. Both the androgenic and spermatogenic functions of the testes are always compromised in Klinefelter syndrome but it is the extent or severity of the androgenic compromise that, for the most part, determines the presentation. Some 47,XXY males may present in teenage years with failure to virilize, their Leydig cells are so impaired and testosterone production so poor, that virilization at the expected time of puberty does not occur. However, if the androgenic deficiency is not as severe, the patient may virilize adequately, escape detection, and only present later in life when infertility and azoospermia lead to the proper diagnosis. Effectively, all men with a pure 47,XXY karyotype will be azoospermic. Approximately 50% of these men may have sperm found upon TESE. A karyotype may also reveal the rare 46,XX male syndrome, in which typically only a tiny portion of the distal aspect of the short arm of the Y chromosome containing SRY is translocated to one of the X chromosomes. SRY is one of the genes involved in the cascade that drives the bipotential gonad in the testicular direction during embryogenesis. The phenotype is male. However, since the majority of the Y chromosome is absent, all of the aforementioned AZF regions are absent and the testes will be devoid of any level of spermatogenesis. This karyotypic finding is prognostic in that TESE is not indicated. In just these first two examples, it is clearly seen that a karyotype should be performed prior to any surgical intervention – it may help make the diagnosis and may help direct therapy. A translocation is discovered in approximately 1-3% of cases (slightly higher in severe oligospermia), and may be the cause of the spermatogenic deficiency and preimplantation diagnosis applied to any embryos created through ICSI may help optimize pregnancy rates. There are other rare findings such as ring Y chromosome and various mosaic states that may also be seen on karyotype. In summary, once the clinical diagnosis of NOA is entertained, it is necessary to obtain both a Y chromosomal microdeletion assay and karyotype before moving to any surgical intervention. There are at least two genetic etiologies for CBAVD. The most common involves the Cystic Fibrosis (CF) genes located on chromosome 7 (80% of cases of CBAVD). Clinical CF is characterized by obstructive pulmonary and pancreatic disease secondary to thick, inspissated secretions. This is the consequence of a poorly functional pool of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, the product of the CF genes. When “severe” mutations occur in both maternal and paternal alleles, clinical CF will result. CF males also have bilateral absence of the vas. However, if the combination of inherited mutations is less “severe” in nature, only vasal absence may be manifest. These men have CBAVD – a clinical condition in which pulmonary and pancreatic function are adequate but the vasa are absent. Of course, there are those men in between these two ends of the spectrum who may have some respiratory tract disease (i.e. sinusitis, bronchitis) or pancreatic disease which is only recognized in retrospect when the diagnosis of CBAVD is made and CF mutations are found to be present. In accord with The American Urological Association Best Practice Policy “Report on the Evaluation of the Azoospermic Male”, “At a minimum, genetic testing for CFTR mutations in the female partner should be offered before proceeding with treatments that utilize the sperm of a man with CBAVD. If the female partner tests positive for a CFTR mutation, the male should be tested as well. If the female partner has a negative test for CFTR mutations, testing of the male is optional.” However, a case can be made that all men with CBAVD require CF mutation analysis prior to sperm aspiration and ICSI. It is necessary to determine if the partner is a carrier of a CF mutation (1 in 25 people of Northern European decent). If she is, preimplantation genetic diagnosis can be used to identify ICSI created embryos that do not carry two mutations and that will not lead to a child afflicted with clinical CF. For the male, he may be the first in his family to demonstrate that CF mutations are present and may have been transmitted to his siblings as well. Family screening and counseling are best done in conjunction with a clinical geneticist. The second presumed genetic etiology for CBAVD involves differentiation of the mesonephric ducts during the first seven weeks of gestation. These men will not have CF mutations and may have unilateral renal agenesis along with CBAVD. The couples need to be informed that there is a small but definite risk of fetal bilateral renal agenesis so that appropriate early level three ultrasounds are performed during pregnancy.

To date, only the above tests are clinically available. However, new etiologies for NOA are being discovered by research labs around the world. Mutations in NR5A1, which encodes steroidogenic factor 1, have recently been detailed in several men with spermatogenic compromise and may provide an explanation in up to 4% of NOA men. Even though the AZFc region may be structurally intact, there may be mutations or functional deficiencies of the many contained genes that reside within that region. In the 4 future, it will be wonderful if it is found that many cases of spermatogenic failure are autosomal recessive or, in various other ways, cannot be passed directly along to the next generation and will, therefore, not result in disease or reproductive insufficiency. This will be of great comfort to our couples as long as we have at least a few spermatozoa to use to help them achieve pregnancy.

Vasovasostomy and Vasoepididymostomy – 2015 Sheldon H. F. Marks, M.D.

Microsurgical vasectomy reversals remain the most cost-effective approach to fathering children after vasectomy, occurring in 2 to 6 % of men after vasectomy. In experienced hands at a specialty center, utilizing state-of-the-art microscopic techniques, success can be as high as 97 to 99.5% for a vasovasostomy and up to 90% for a vasoepididymostomy. Reversals are well tolerated with minimal risks, often with a rapid return of fertility. The microsurgical skills required for successful reversals are difficult to perfect, rapidly perishable and require frequent and regular practice. Simply knowing what to do is not enough. Reversals are not for the “occasional” microsurgeon.

Predicting success Predicting success is difficult and cannot be done accurately for an individual pre-operatively. Even a good physical exam and detailed history is suggestive at best. Most experts agree success is primarily dependent on the skill and expertise of the microsurgeon. The highest success rates with patency up to 99.5% with the fewest complications are achieved by the most experienced. Another key factor is the intra-operative interpretation of the vasal fluid, both gross and microscopic, which directs whether a VV or VE is the correct procedure. The goal of a reversal is > 10 million motile sperm in the ejaculate. Pregnancy is not a good endpoint for measuring reversal success. If patient has good sperm numbers post-reversal, conception is primarily dependent on female age/factors. There are many independent female and other male factors beyond the technical reversal, pre and post-reversal, that impact on reversal success. These include female age, health, weight, male health, medications, testosterone replacement therapy, lifestyle choices and prior fertility, individually and together, to name a few. A frequent misconception persists that the number of years from vasectomy is an important variable for predicting success. We do know that statistically, the longer the interval since vasectomy, the more likely the pressure within the epididymal tubules will exceed capacity, resulting in epididymal blow- out, obstruction and so necessitating a VE. The likelihood of this epididymal obstruction increases linearly and plateaus at 22 years and beyond, with about 1/3 of men requiring bilateral VE’s, 1/3 VV/VE and 1/3 bilateral VV’s. For an individual patient, we have seen the need for VE as soon as 2 years from vasectomy and favorable fluid with a VV in men as far out as 30 to 42 years from vasectomy. There is no obstructive interval at which the surgeon should assume the patient should automatically receive VV’s. The microsurgeon should be ready, trained and prepared to perform a VE or VV any number of years from vasectomy. Another common myth with reversal success is potential impact of antisperm antibodies (ASA) on post-reversal fertility. Though present in many men with variable and inconsistent patterns, these antibodies have been shown to be a non-issue for the vast majority of reversal patients. Even patients with 100% antibody binding are able to conceive children naturally. Despite the rarity of clinically significant antibodies, sperm autoantibodies are incorrectly discussed as a contraindication for reversal on the Internet and promoted by the uninformed.

Anesthesia options Most common choices for vasectomy reversal are general anesthesia or conscious IV sedation with local. The key is to keep the patient comfortable and provide a steady surgical platform. The anesthesia technique you choose depends primarily on surgeon preference. Other factors include patient request, reversal anatomy specific issues such as whether the vasectomy was trans-scrotal or inguinal, deep pelvic or an anticipated complex case or challenging redo “salvage” reversal. General anesthesia allows the surgeon to move a bit faster with the patient asleep. Concerns with general anesthesia are rare risks, potential for incomplete cord blocks, increased costs and post- anesthesia nausea and vomiting – not desirable in a fresh post-reversal patient. If you opt for conscious IV sedation, which is my preference, most use low dose Fentanyl and Versed, continuously monitored, with local cord blocks. Giving the patient a handful of multiple oral medications alone pre-op is not good practice.

Microsurgical equipment techniques/principles Always use the right tools for the right job. Cutting corners and using cheaper equipment, 5 microinstruments, microsutures and supplies often leads to poorer quality results, more frustration and lower success. Look at various micro-tipped instruments to find ones that fit your needs. You are encouraged to talk to experts for their recommendations for instrument and suture manufacturers that consistently provide quality products. When considering microsurgical instruments, cheap is cheap for a reason. Poor quality instruments slip, are not precise, malfunction and break. Top quality instruments are a better value, last longer and are worth the cost. Many instruments labeled as “micro” are not as fine tipped as labeled, so check them out. Micro-tipped forceps, needle holders and microscissors should all be non-locking and shave round rather than flat handles to allow for more precise rolling of the instrument between the fingertips. The microsutures themselves are critical to successful reversals. There will always be less expensive sutures available. If achieving the highest level of success is important, then you should always use the best 10-0 and 9-0 non-absorbable nylon microsutures. It is very important to use the correct tissue-specific, task-specific needle, such as those expressly engineered and manufactured for the various layers of a vasovasostomy or vasoepididymostomy. Most experts prefer a 70 or 100 micron bicurve needle for the mucosal and muscularis layers such as the MET brand by Surgical Specialties. A generic cutting needle will cause too much tissue damage, as can a standard tapered needle where extra force is required to push through the tissue. Precise needle placement is very important. If the needle pass is hesitant or too forceful, then you risk increased trauma to delicate tissues with increased inflammation and scarring. It should be a single, smooth pass of the needle, “rolling” the needle into and through with subtle twist of the fingers. Avoid jerky motions. Grasp mid-needle with the micro-needle holders for the best control. Incorrect holding or passing the microneedle can cause it to bend – it is not the needle that is flawed. Do not grab the point of needle after it is passed through, as this dulls or bends the tip. Tie sutures down with “finesse” – not too tight, not too loose – “just right,” which comes with practice. Your choice of a surgical microscope is important. You will achieve the best results when you use a top-of-the-line double headed surgical microscope (Leica, Zeiss) with XY axis foot control. It is always much easier on your eyes during a several hour case when you use better optics for better visualization, especially under higher magnification as needed for challenging surgeries and precise placement of sutures into the epididymal tubule. Avoid using a cheaper training microscope.

Microsurgical techniques/principles The key concepts of a successful anastomosis that we all learned in general surgery still apply: the anastomosis must be tension-free, have a good blood supply to both proximal and distal ends, a watertight repair, no abdominal obstruction (such as from prior hernia repair) and an adequate, open lumen upon completion. Know and meticulously preserve the vasal blood supply- don’t be cavalier tying off or cauterizing blood vessels unless necessary. Do not skeletonize the vas – it poor form to try for the vas to look pristine, white and dry. Rather, we want to see some oozing from transected ends, verifying good blood supply, so critical for healing. If the vas is aggressively skeletonized, then increase the risks for ischemic necrosis of the vas and vasal repair, manifest as delayed anastomotic obstruction. Avoid cautery near the vas. Bipolar only near vas but minimally. Not just at the transected face and mucosa but along entire vasal length. Maintain meticulous hemostasis. Control, tie or bipolar small vessels to reduce small micro-hematomas or blood dissecting along the cord. Avoid absorbable sutures adjacent to the vas. Transection of the vas should be performed with a clean, single, smooth pass with a new microblade. It is important to use a fresh microblade as they noticeably dull after just a few passes. Transection should be a smooth, steady, slicing motion. Avoid sawing back and forth. Use of the Marks angled Vas Cutting Forceps (ASSI # NHF-2.5.15) will reduce shearing trauma seen with blunt transection and increase anastomotic patency by providing for a larger lumen with up to 99.5% success as compared to the standard 90 degree forceps. A central, round lumen with “bulls eye” appearance is the goal. If you see an off-center lumen in the deep convoluted vas or if evidence of scarring in the muscularis of the vas, you may need to transect at 0.5 millimeter intervals (use a new blade) to find an ideal round lumen. Inspect and verify a healthy, cut mucosa. Follow a “no-touch” technique to the transected face of vas. To minimize trauma when you need to manipulate the vas, pick up loose peri-vasal adventitial tissues several mm away from transected face. Do not forcibly dilate the lumens with lacrimal duct dilators, which can be very traumatic with increased risks for scarring. Instead, you can use the tip of the micro-forceps, being very gentle just enough to see the lumen mucosa. Minimal traction on the contralateral adventitia will help to visualize the lumen edges. A skilled assist can make any reversal much easier. The assist should stay proactive, always thinking and preparing one or two steps ahead of the surgeon. 6

The incision Most use a single vertical midline or paramedian vertical scrotal incisions. There are pros and cons to each. Your decision will be based on the individual patient’s anatomy, the size and location of the vasal defect, prior scrotal or inguinal surgery, the abdominal vas location, the likelihood for needing to extend the incision for a more proximal dissection or repair, and finally, the most commonly used reason for incision location is surgeon preference. You will need inguinal incisions for an inguinal reversal. Micro- incisions can be used.

Gross fluid evaluation It is important to characterize the gross fluid findings from vasal end and record for each side. These gross findings often correlate with the microscopic findings and may be predictive of whether you will need to perform a vasovasostomy or vasoepididymostomy. You should not use gross fluid findings alone to decide whether to preform a VV or VE. Each side is an independent system and so you can frequently find different fluid on one side from the other. Fluid categories most often used include color (clear, murky, yellow, brown and white), consistency (watery - the best, creamy, oily, thick and the worst is toothpaste-like), and volume (which can vary widely from none, minimal, mild, moderate to copious). Volume can change from minimal initially and become moderate to copious later into the repair.

Microscopic fluid evaluation Looking at the vasal fluid under a bench microscope (400X) during the reversal is the most important factor to decide whether a VV or VE is indicated and the best predictor of post-reversal fertility. You should have a high quality, high power, phase-contrast microscope in the OR for the surgeon to review the slides or have the vasal fluid analyzed real-time by an andrologist. Here in Arizona at ICVR, we have our full-time andrology lab adjacent to our operating suite. After the surgeon looks for sperm with the OR microscope, the slide is handed to our andrologist who performs a more comprehensive analysis of the slide to look for and quantify the presence and quality of sperm or parts. A monitor located in our O.R. allows the surgeon to see what the andrologist sees on the lab scope. It is important to document whether there are whole sperm (as well as the count/cc, % motility and forward progression), sperm with partial tails (short or long tails), sperm heads, sperm tails, snow (debris), and finally if the fluid is bankable for IUI or IVF/ICSI. If no sperm are seen, analyze a 2nd or even 3rd slide, with gentle “no pressure” heparinized saline barbotage if needed, as sperm can appear in subsequent slides. Banking is offered, when available, at the discretion of the patient. If sperm are seen and so there is evidence of testis to vas continuity, then perform a vasovasostomy. If there is no sperm in the vasal fluid, which is consistent with deeper epididymal obstruction, then a vasoepididymostomy indicated.

Prepping the vas First identify and transect the vas in what appears to be normal vas above and below the vasectomy site. The goal is to find healthy, “bulls-eye” appearance of the transected vas. Microdots, as described by Dr. Goldstein, are then placed, ensuring accurate placement of the sutures to maintain orientation. Use the micro-tipped marking pen to outline mucosal edge to assist with precise suture placement. If use angled vas cutting forceps, place double dots at 12 o’clock. When prepping the abdominal vas, place the microdots first. Then perform a hydraulic pressure vasogram by slow, gentle instillation of 1 to 2 cc of heparinized saline via 24 g Angiocath sheath. Do not blast it through. You are looking for easy flow as presumptive evidence of abdominal vasal patency.

Vasal set up and stabilization This is perhaps the most critical (and often challenging) portion of the reversal. If set up correctly, the reversal becomes much easier. It is essential to secure and align the vas proximally and distally while maintaining good blood supply. Inadequate set-up creates an ever more challenging procedure with a higher likelihood for problems, compromised technique and failure. The vas should be on no tension, with the lumens “kissing.” The most common techniques to achieve this include 1. use of a Goldstein Microspike Approximator Clamp (ASSI # MSPK-3678), 2. adventitial holding stitches, 3. securing the vas onto a tongue blade inside of Penrose drain and 4. securing the vas through small holes in an instrument wipe. The latter offers the benefit of providing a white back-drop which of benefit while working with the extremely fine 10-0 sutures.

Vasovasostomy multilayer technique There are other techniques, such as the modified 1-layer and 2-layer, but most top experts prefer the multilayer which provides the most anatomic repair. Initially place and tie three posterior 9-0 nylon stitches to secure the posterior muscularis. Then place and tie the first of three interrupted 10-0 nylon 7 inner layer sutures at 6 o’clock, then 4 and 8 o’clock position. When placing these inner layer mucosal sutures, it is important to include about 1/3 thickness of the muscularis and edge of the mucosa with each bite. Be consistent with your technique. Be sure the knot is not within the lumen. Verify after each suture is placed that the opposite mucosal wall has not been inadvertently included in the stitch, which would effectively seal the lumen shut. Once completed, place but do not tie the three front wall mucosal stitches. Gently irrigate the lumen with heparinized saline to remove clots and debris, and then tie the front wall mucosal sutures. Place and tie the remaining muscularis sutures of 9-0 nylon to reinforce and add strength to repair. Each interrupted suture should include 1/3 to ½ of the muscularis and be 1 mm apart circumferentially. The thin walled convoluted vas is more challenging. Finally, approximate the outermost adventitial layers with interrupted 7-0 nylon sutures. The number of peri-vasal adventitial layers, whether one or two, is anatomy dependent.

Vasoepididymostomy intussusception technique Few other surgical procedures are so dependent on the surgeon’s technical expertise. The number of years from vasectomy is only suggestive at best of the technique that may be needed. We have seen men 2 years from vasectomy with epididymal blowouts requiring bilateral VE’s. If you start a reversal you should be ready, trained and capable of performing a VE. It is important to free up the abdominal vas proximally preserving the peri-vasal adventitia. It is very difficult perform a VE with limited proximal dissection. With the testicle delivered and tunica vaginalis opened, pass a fine hemostat out through the most superior tunica vaginalis, grab the vasal adventitia and draw the abdominal vas through the tunical defect. Secure the vas at multiple points with 7-0 nylon sutures. Positioning the vas up to the epididymal tubule can be the most challenging and most time consuming aspect of a VE. The goal is to have the transected abdominal vas aligned with the open epididymal tubule. You may need to consider a “relaxing incision” in mesentery of epididymal tunic to allow for reduced tension on anastomosis. Reduce the micro-bipolar to low setting. Try to attach the abdominal vas to the most distal epididymal tubule containing sperm to allow for as much epididymal maturation as possible. Once you have found the target tubule, score the tunica over and parallel with the tubule with Lipshultz Microdissection Scissors (ASSI # SDC-15R VL) and then gently under high magnification tease off tissue from the tubule. To open the tubule, either excise a tiny longitudinal ellipse in the chosen tubule with microscissors and pass the 10-0 needles along either side of the defect or incise with a 15 degree ophthalmic blade between placed 10-0 needles. Then secure the back wall of the vasal muscularis to inferior edge of the tunic with 3 interrupted 9-0 nylon sutures. We use the 2 suture end-to-side longitudinal intussusception technique with double armed 10-0 nylon sutures on 70 micron bicurve MET needles (Surgical Specialties # 2492). This is an easier technique with excellent success. Once the sutures are placed, the tubule is drawn up into the vasal lumen and sutures tied. On occasion, you may need to perform the classic 5 or 6 point end-to-side approximation. Complete the anterior muscularis/tunica layer using interrupted 9-0 nylon stitches placed 1 mm apart. Then reinforce the adventitia/tunica layer with interrupted 7-0 nylon as needed, close the tunica vaginalis and replace the testicle.

Sperm granuloma The presence of a sperm granuloma on one or both sides is considered a very favorable finding, with good prognostic implications as it predicts the absence of a deeper epididymal obstruction. Granulomas can be tender to very painful, though many patients are unaware of the granuloma. These act as a pressure release “valve,” preserving sperm function in a low-pressure state. When the vas is transected just below the granuloma, you usually see no lumenal dilation. The classic fluid findings with a granuloma, though not always, include minimal volume of clear, watery fluid with occasional to very rare whole, often motile sperm. It is best to routinely excise granulomas rather than leave this inflammatory lesion adjacent to your repair.

Post-operative care A ¼ inch Penrose drain is rarely needed but may be used overnight, based on surgeon preference and perceived need such as with increased drainage often seen after a difficult redo salvage reversal. Recommend that the patient use ice packs, intermittently, for the first 48-72 hours to minimize swelling and discomfort with strict “house arrest”. We ask that the patient limit himself to light duty during the next 14 days and wear an athletic supporter 24/7. It is important for the patient to abstain from ejaculation for 14 days, with no heavy lifting for 4 to 6 weeks. In our practice we prescribe NSAID’s for 4

8 to 6 weeks. If a redo reversal or bilateral VE’s, we use a course of tapering prednisone followed by NSAID’s. The patient is instructed to check his 1st semen analysis at 4-6 weeks, with follow-up counts regularly every Q 4 to 6 weeks for 6 months (compliance is a challenge). If SA results are suboptimal or less than we would have predicted based on intra-operative findings and the specific technique, then we initiate our anti-inflammatory protocol (tapering prednisone/NSAID’s) and recheck SA in 4 to 6 weeks.

Intra-operative dilemmas There are a multitude of intra-operative dilemmas that regularly present unexpected challenges and frustration even for the most senior, experienced microsurgeons. These include discrepant lumens, lengthy vasal gaps, pouchy mucosa, deep convoluted vas - almost to the epididymis, vasitis nodosa and a very high, often fixed abdominal vas with need for VE. Indeterminate vasal fluid is one of the most common dilemmas, as well as thick vasal fluid suggesting epididymal blow-out yet the epididymis flat with nondilated, nonvisible tubules, or thick, creamy fluid suggestive of a blow-out yet with occasional sperm seen microscopically. Our general surgery colleagues provide us with some of the most challenging scenarios – an inguinal vasectomy or compromise of the vas from direct damage or adjacent mesh at time of inguinal herniorrhaphy or a deep pelvic vasectomy or damage during another laparoscopic procedure, most commonly lap herniorrhaphy. These cases are always extremely challenging, often lengthy with unknown outcomes. It is always important to explain to patients your concerns over the potential for your inability to perform a reversal with other options to include laparoscopic robotic exploration with repair or IVF/ICSI.

Redo “salvage” reversals Redo reversals (and even redo, redo reversals) can have a high success rate. In fact, redo reversal results at our Center are equivalent to first time reversal rates. Redo reversals can be very difficult, extremely time consuming often with extensive, dense peri-vasal scarring and even tunical obliteration and scarring, which often depends on the extent of the first surgeon’s technique. Redo reversals require slow, meticulous dissection. In our practice we usually anticipate an additional hour of surgery time or more. When performing a redo, the surgeon should expect the unexpected.

Peter N. Schlegel, MD Sperm retrieval techniques

The development of methods of micromanipulation of gametes in the laboratory has improved the results of in vitro fertilization (IVF) when only very small numbers of sperm are available or when sperm motility and morphology are abnormal. Intracytoplasmic sperm injection (ICSI) truly has revolutionized the treatment of infertile men since its introduction in 1992. The ICSI procedure requires only a single sperm to be injected into each viable ovum produced in a standard IVF cycle, potentially permitting genetic pregnancy in even the most severe cases of male infertility, including: (1) azoospermia (complete absence of sperm in an ejaculated specimen – either obstructive or non-obstructive); (2) cryptozoospermia (no sperm seen on routine analysis, but rare sperm found in many centrifuged microscopic fields by the laboratory technologist); (3) retrograde or anejaculation; and (4) no morphologically normal sperm present. Many IVF centers in the U.S. and abroad routinely report pregnancy rates ranging from 35 – 55% in couples with severe male factor infertility, many of whom had little if any chance at genetic pregnancy previously. The availability of ICSI spurred the development of a myriad of techniques used to harvest and cryopreserve sperm in men with azoospermia, whether related to obstruction (e.g. previous vasectomy) or severe testicular dysfunction (e.g. genetic infertility). The proper clinical application of these techniques demands a thorough understanding of the diagnosis and evaluation of the azoospermic male, close cooperation with an assisted reproductive laboratory, and a keen sensitivity to the psychological and financial concerns of the involved couple.

Requirements for Sperm Retrieval The IVF laboratory must have ICSI expertise, and laboratory personnel should optimally be present during the sperm retrieval procedure, whether that site is in the office, operating room or IVF facility. The same microsurgical instruments used for vasovasostomy and vasoepididymostomy may be used for open sperm retrieval methods, and instruments useful for percutaneous retrieval methods are discussed subsequently. A sperm nutrient solution, such as human tubal fluid (HTF), should be supplied by the laboratory personnel. Serologic testing for Hepatitis B, Hepatitis C, HIV, and syphilis, are recommended for testing of sperm donors before cryopreservation and are required by most IVF laboratories before sperm retrieval. A laboratory microscope must be available during the operative procedure for the assessment of the numbers and motility of sperm obtained . Open sperm retrieval methods typically require the availability of an operating microscope. 9

Retrieval Methods The sperm retrieval methods are: MESA = Microsurgical Epididymal Sperm Aspiration PESA = Percutaneous Epididymal Sperm Aspiration TESE = TEsticular Sperm Extraction TESA = TEsticular Sperm Aspiration (percutaneous)

Percutaneous Testicular Biopsy (Needle biopsy) Sperm may be retrieved either from the epididymis or from the testicle in patients with obstructive azoospermia (OA) with equally good success rates in most laboratories. However, testicular sperm is required for ICSI when patients have nonobstructive azoospermia (NOA). A published meta-analysis has confirmed the widely held belief that IVF success is lower when using testicular sperm from men with NOA than when using testicular sperm from men with OA. MESA (Fig. 20) involves exposure of the epididymal tubule, creation of a microsurgical opening into the tubule and subsequent aspiration of the tubular fluid into 1 cc syringes containing small volumes of sperm nutrient solution. A small (22-27 gauge size) Medicut catheter or angiocatheter may be attached to the syringe to perform the aspiration. It is useful to depress the area of the opened epididymal tubule in order to create a well from which the epididymal tubular fluid can be aspirated. Alternatively, some centers have used a micropuncture approach that allows collection of microliter and larger volumes of epididymal sperm using a specially produced glass micropipette. Epididymal sperm obtained in this manner are only suitable for use with IVF/ICSI. Typically, the laboratory is instructed to freeze the sample into multiple tubes or straws prior to cryopreservation, eliminating the need for any future retrieval procedures for the male partner. When performing MESA, a “reversed motility” of sperm may be found. Contrary to the situation in the unobstructed epididymis, it may be necessary to obtain epididymal tubular fluid from the highest levels of the epididymis or even from the efferent ducts in order to obtain motile sperm from the obstructed epididymis. The epididymal dissection is therefore initiated at the caudal end and carried proximally until high quality motile sperm are located. The laboratory technologist should be able to perform rough sperm count estimations during the MESA procedure to help the surgeon decide when a sufficient number of sperm has been obtained. Ideally, each aliquot would contain 250,000 to 500,000 motile sperm. If there is a disparity in testicular size, MESA is performed first on the side of the larger testicle. Occasionally, it is necessary to perform MESA bilaterally in order to obtain a sufficient number of sperm for cryopreservation in enough aliquots for future IVF/ICSI cycles. When the MESA procedure is completed, some surgeons close each incision into the epididymal tubule microsurgically, while other surgeons simply cauterize each incision in order to seal it closed. PESA is performed using a 21- to 25-gauge butterfly (scalp vein) needle attached by a length of intravenous tubing to a 1 cc tuberculin syringe. The needle is inserted into the epididymis, the plunger of the syringe is pulled to create negative pressure until a column of epididymal fluid appears in the tubing, which then is clamped between the fluid and the syringe before removing the needle from the epididymis. The use of PESA with IVF has reported similar pregnancy rates to those obtained using ejaculated or microsurgically retrieved sperm. Whether PESA or MESA provides better success rates with IVF-ICSI has not been tested in a controlled study. Since PESA samples mix older sperm with higher DNA fragmentation, it is possible that PESA may provide sperm with more abnormal DNA integrity. Irreversible epididymal injury may occur after PESA,precluding a future reconstruction if desired. Lisek and Levine discussed technical aspects of performing PESA. TESA is performed with a 21 gauge 1½ inch long needle attached to a 20 cc syringe, which is placed in a Cameco type of pistol grip apparatus available from Precision Dynamics Corp (see section on INSTRUMENTS). Maximum negative pressure is created, following which the needle is moved in and out in a single plane within the testis multiple times. Before withdrawing the needle, the negative pressure is released slowly to prevent the aspirated tubule from returning into the testicle. After the needle is withdrawn, it is detached from the syringe by the laboratory technologist. Air (5 to 10 cc) is aspirated into the syringe, the needle is reattached and the air is used to force the contents of the needle into the sperm nutrient solution by depressing the plunger of the syringe rapidly. TESA also may be performed using a Biopty gun with an 18 or 20 gauge needle. Another method of performing TESA using a 16 gauge intravenous catheter with attached intravenous tubing is described by Marmar and by Fahmy et al, who use this technique to obtain large enough quantities of testicular tubule to allow cryopreservation. After each needle puncture of the testis, firm pressure is maintained over the puncture site for 1-2 minutes to ensure hemostasis. Percutaneous testis biopsy can also be carried out using a short-excursion needle biopsy gun similar to that used during prostate biopsies, to obtain small volumes of testicular tissue that typically have larger numbers of sperm than are obtained with TESA. Typical prostate needle biopsy cores are taken with an 10 18 gauge needle that has a 17 mm excursion. Since this length may penetrate through the width of the testis, shorter excursion (1 cm) guns are often used, but the bore of these needles is usually wider (15 gauge.) It is not clear if there is any greater risk of bleeding with the larger bore needle. Following TESA (as with PESA), the aspirated tubule is “milked” to express the sperm into small aliquots of sperm nutrient solution. We prefer to use 50 microliters of sperm nutrient solution in an Eppendorf tube. The use of larger volumes of sperm nutrient solution makes identification and isolation of the aspirated sperm more difficult for the IVF laboratory personnel. We found an average of 75% of testicular sperm obtained by TESA from men with obstructive azoospermia to be viable. This accounts for the relatively high fertilization rates using such sperm for ICSI. Most IVF laboratories report pregnancy rates of 40% to 55%, equivalent to success rates achieved with normal ejaculated sperm. TESE involves sperm retrieval from testicular tissue by some form of open biopsy and is the only available option for men with nonobstructive azoospermia who desire genetic offspring. When considering TESE for men with nonobstructive azoospermia, the surgeon should be aware that the results of a diagnostic testicular biopsy may not correctly predict what may be found during an IVF/ICSI cycle. In most centers, pre-treatment diagnostic biopsy is no longer performed, eschewed in favor of combined biopsy and sperm retrieval, either prior to an IVF cycle with cryopreservation, or as an in-cycle procedure at the time of egg retrieval. Schlegel and Su reported the physiologic consequences of performing multiple testicular biopsies for TESE in men with nonobstructive azoospermia, including impaired testicular blood flow in two men, one of whom had complete testicular devascularization after multiple biopsies. An alternative approach that uses a method of “mapping” the testis by multi-site fine needle aspiration to determine the best site(s) to later perform TESE or TESA has been reported by Turek, et al and is gradually gaining wider acceptance. Pre-cycle mapping can be used to attempt to localize sperm production within the testis, but its success at sperm retrieval has not been directly compared to microdissection TESE. In addition, different IVF centers have widely discrepant success in using testicular sperm from men with nonobstructive azoospermia. Prins et al from Chicago report similar success using fresh & frozen testicular sperm from men with NOA, but other centers such as Cornell report low (33%) freeze- thaw viability of sperm and lower pregnancy rates with frozen-thawed testicular sperm even in the few cases where sperm survive. A microsurgical method of TESE (micro-TESE) has been reported by Schlegel to result in obtaining a maximum number of sperm from a minimal amount of testicular tissue in men with nonobstructive azoospermia. He found that seminiferous tubules that appear to be full, or dilated, as contrasted with surrounding collapsed seminiferous tubules, are the tubules most likely to contain sperm. The procedure involves an intensive microsurgical search through the testicular parenchyma. Initially, the testis is opened widely in an equatorial plane to allow exposure of seminiferous tubules. Then, microdissection is performed along tissue planes, preserving the testicular blood supply while allowing exposure of additional areas of testicular tissue. The procedure is terminated when sperm are found or all accessible areas of the testis have been directly examined. The procedure can take several hours to allow examination of all areas of both testes, even in experienced hands. Some investigators have found micro-TESE to be advantageous, while others have not found it more useful to obtain testicular sperm from men with nonobstructive azoospermia than the performance of multiple testicular biopsies without the aid of microsurgical techniques. A survey of urologic microsurgeons who specialize in male infertility from North America indicated that 80% of these experts will use microdissection as part of their treatment of men with non-obstructive azoospermia, although not all use it as initial or primary treatment. Several controlled, peer reviewed published studies have confirmed the superiority of microTESE over multiple random biopsies to extract sperm from men with non-obstructive azoospermia. Some centers have not reported this experience, but it is not clear if their studies were adequately powered to detect the benefit of microdissection TESE, nor if they were performeing the same procedure. Microdissection TESE can be a prolonged tedious procedure since there may be few, limited sites of sperm production in some men with NOA. Studies evaluating patients after microTESE suggest that fewer ultrasound-detected changes (meaning less tissue scarring or damage) occur after microTESE compared with multiple random biopsies of the testis (TESE). Seminiferous tubules that are obtained by TESE or TESA may require mechanical disruption of the tubules or dissection to release spermatozoa from within the tubules into the surrounding sperm nutrient solution. Most IVF laboratories first dissect the tissue between 2 small (26 or 27 gauge) needles. If sperm are not obtained by such dissection, enzymatic digestion of the tissue (with 0.1% collagenase) may be performed to release the sperm from the seminiferous tubules. When only non-motile sperm are obtained during TESE or TESA, the hypo-osmotic swelling (HOS) test or treatment of retrieved spermatozoa with 3 mM pentoxyfilline may be useful to select viable sperm that should be used for ICSI. All sperm aspiration methods, including testicular mapping, may be performed with spermatic cord block with scrotal skin local anesthesia in an office setting. MESA and TESE may be performed with

11 either local or general/epidural anesthesia in an outpatient facility, while micro-TESE typically requires regional or general anesthesia.

Timing of Retrieval When deciding which type of sperm retrieval best suits both the infertile couple and the operating surgeon, it is vitally important to ask the involved IVF team/lab personnel what “type” (source) of sperm they prefer. Ideally, all IVF labs should be facile at handling both epididymal and testicular sperm, whether from fresh or frozen preparations. In individual labs, one source may be preferred and should be provided if possible. In cases of obstructive azoospermia, nearly all centers report similar lVF success using any sperm source, allowing for the use of any of the described techniques. The use of cryopreserved testicular sperm obtained by TESE for later IVF/ICSI has been reported by several groups. The concept of a therapeutic testicular biopsy, i.e. a diagnostic biopsy and simultaneous testicular cryopreservation, is utilized at many reproductive centers with overall success similar to that achieved with testicular sperm. Similarly, MESA allows the convenience of pre-cycle scheduling and cryopreservation while providing the lab with large numbers of easy-to-handle epididymal sperm specimens. Both MESA and TESE are a bit more invasive, and when performed with regional/general anesthesia, each may increase morbidity and out of pockets costs to the couple. When using PESA and TESA, the procedure is typically performed on short notice and a relatively small number of sperm are obtained. However, both may be performed with local anesthesia in the office, which significantly reduces overall costs and morbidity. PESA sperm may have higher DNA fragmentation than testicular sperm because PESA sperm often are admixed with macrophages or derived from more “distal” regions of the epididymis. In general, centers that use PESA have lower pregnancy rates, but it is not clear if these differences are because of the center or specific differences in sperm quality. In cases of non-obstructive azoospermia, testicular sperm retrieval is required, and may be performed fresh or pre-cycle with cryopreservation. A published meta-analysis has demonstrated inferior IVF results when using frozen/thawed testicular sperm from men with NOA. It should be noted that even investigators who report similar fertilization and pregnancy rates with frozen/thawed testicular sperm from obstructed patients note lower fertilization and pregnancy rates with frozen/thawed than with fresh testicular sperm obtained for ICSI from men with nonobstructive azoospermia. Some centers now favor the use of fresh, rather than frozen/thawed, testicular sperm for IVF/ICSI. Unfortunately, 35-45% of couples will undergo ovarian hyperstimulation only to find that no sperm are present on the day of egg retrieval, necessitating the use of backup donor sperm for the IVF cycle. Some centers approach this problem by performing microTESE on the day prior to oocyte retrieval of the female partner. If no sperm are found, the egg retrieval is cancelled, although stimulation was already done. To minimize the number of “unnecessary” IVF cycles, other centers have used diagnostic testicular mapping to predict which men with NOA will have retrievable sperm PRIOR to the IVF cycle. It is not clear how many men with “negative maps” could have sperm retrieved with microTESE.. At the centers offering this approach, patients with positive maps are offered IVF with in cycle fresh retrieval, using either TESA (if diffusely positive) or micro-TESE (if focally positive). We will consider patients for sperm retrieval with microTESE if prior TESA/FNA failed to retrieve sperm, as noted below.

Prognosis: Chance of sperm retrieval The chance of sperm retrieval for men confirmed to be azoospermic at Cornell on the day of planned sperm retrieval (and using a detailed microTESE procedure )has been outlined in a series of publications. For men with Klinefelter syndrome, the chance of retrieval is 68% per attempt, with AZFc deletions, 70%, for men with cryptorchidism and prior orchidopexy, 74%, with idiopathic azoospermia, 50%, with a history of prior chemotherapy for germ cell tumor, 83%, for chemotherapy with alkylating agents, 33%, for men with diffuse maturation arrest 40%, and with Y chromosome microdeletions involoving all of AZFa or AZFb, 0%. Prior failed biopsy does not significantly affect sperm retrieval, but if 3 or more biopsies per testis failed, then the chance of microTESE drops to 10-20%, depending on the histologic pattern observed during attempted TESE. Pre-treatment hormonal therapy (with clomiphene citrate or aromatase inhibitors[e.g., anastrazole or letrozole]) may optimize the chance of sperm retrieval as well as to increase circulating testosterone levels.

New Genetic Tests Spermatozoa can be used with IVF-ICSI, and until recently, as long as the sperm are viable, no sperm characteristic has been thought to adversely affect ICSI results. A series of studies have been done to evaluate the effects of sperm DNA (DNA integrity and aneuploidy) on IVF-ICSI outcomes. Although fertile men rarely, if ever have abnormal sperm DNA integrity, high levels of abnormal DNA integrity are found in infertility patients. Men with abnormal sperm DNA integrity or sperm aneuploidy 12 (abnormal complement of chromosomes in individual sperm) are more likely to contribute to pregnancies even with IVF-ICSI, and even after pregnancy is achieved, they are more likely to have a spontaneous miscarriage. Some have also suggested an increased risk of childhood cancers when sperm DNA fragmentation abnormalities were present. It does not appear than abnormal sperm DNA integrity affects fertilization rates, but later embryo development, implantation, and maintenance of pregnancies is adversely affected. Sperm DNA integrity is measured by SCSA or TUNEL analyses to detect sperm DNA breaks. It appears that both SCSA and TUNEL have similar predictive value in determining the outcome of ICSI attempts, although absolute values may differ slightly from these tests. In both cases, threshold levels of abnormal sperm DNA are 27-30%. When more than 30% of sperm have abnormal DNA integrity, results of IUI, IVF and ICSI are adversely affected, according to a meta-analysis published by Collins et al in Fertility & Sterility. Treatment of abnormal DNA integrity with antioxidants, varicocele repair (for men with abnormal sperm DNA & varicoceles), and testicular sperm retrieval have been attempted for men with abnormal sperm DNA integrity in ejaculated spermatozoa. Although better sperm DNA fragmentation is found in testicular sperm, there is not a definite fertility benefit when using sperm from the testis. It is not clear what clinical conditions are associated with sperm aneuploidy, nor what treatments are possible for abnormal aneuploidy, although one report suggested decreased sperm aneuploidy after varicocelectomy. Although both sperm DNA abnormalities (aneuploidy & abnormal DNA fragmentation) can adversely affect ICSI success, there is no cutpoint of abnormalities that precludes success of ICSI, so evaluation of patients before attempted treatment is not always clinically necessary.

References for Evaluation and Genetic Basis of Azoospermia 1. Report on evaluation of the azoospermic male. Fertil Steril 2008;90 (5 Suppl):S74-7. 2. Zhang F1, Li L, Wang L, Yang L, Liang Z, Li J, Jin F, Tian Y. Clinical characteristics and treatment of azoospermia and severe oligospermia patients with Y-chromosome microdeletions. Mol Reprod Dev. 2013 Nov;80:908-15. 3. McLachlan RI, O'Bryan MK. Clinical Review: State of the art for genetic testing of infertile men. The Journal of Clinical Endocrinology and Metabolism; 2008; 95:1013-24. 4. Oates R, Lamb D. Genetic aspects of infertility. In: Lipshultz L, Howards S, Niederberger C, eds. Infertility in the male. 4th ed. New York: Cambridge University Press; 2009:251-76. 5. Navarro-Costa P, Plancha CE, Goncalves J. Genetic dissection of the AZF regions of the human Y chromosome: thriller or filler for male (in)fertility? Journal of Biomedicine & Biotechnology; 2010; 93:65-69. 6. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 2003; 423:825-37. 7. Repping S, Skaletsky H, Lange J, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. American Journal of Human Genetics 2002; 71:906-22. 8. Stahl PJ, Masson P, Mielnik A, Marean MB, Schlegel PN, Paduch DA. A decade of experience emphasizes that testing for Y microdeletions is essential in American men with azoospermia and severe oligozoospermia. Fertil Steril 2010; 94:1753-6. 9. Rozen SG, Marszalek JD, Irenze K, Skaletsky H, Brown LG, Oates RD, Silber SJ, Ardlie K, Page D: AZFc deletions and spermatogenic failure: a population based survey of 20,000 Y chromosomes. Am J Hum Genet 2012 91:890-6. 10. Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 2002; 17:2813-24. 11. Aksglaede L, Link K, Giwercman A, Jørgensen N, Skakkebæk NE, Juul A. 47,XXY Klinefelter syndrome: Clinical characteristics and age-specific recommendations for medical management. Am J Med Genet (C) Semin Med Genet 2013; 163C: 55–63. 12. Oates RD. The natural history of endocrine function and spermatogenesis in Klinefelter Syndrome. Fertil Steril. 2012 Aug;98(2):266-73. 13. Kristian A. Groth, Anne Skakkebæk, Christian Høst, Claus Højbjerg Gravholt, and Anders Bojesen Clinical Review: Klinefelter Syndrome—A Clinical Update JCEM 2013 98: 20-30. 14. Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod 2003; 18:1660-5. 15. Oates RD, Amos JA. The genetic basis of congenital bilateral absence of the vas deferens and cystic fibrosis. J Androl 1994; 15:1-8. 16. Ratbi I, Legendre M, Niel F, et al. Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling. Hum Reprod 2007; 22:1285-91.

13 17. Schwarzer JU, Schwarz M. Significance of CFTR gene mutations in patients with congenital aplasia of vas deferens with special regard to renal aplasia. Andrologia 2012 44: 305-307. 18. Gatta V, Raicu F, Ferlin A, et al. Testis transcriptome analysis in male infertility: new insight on the pathogenesis of oligo-azoospermia in cases with and without AZFc microdeletion. BMC Genomics 2010;11:401. 19. Hotaling JM. Genetics of Male Infertility. Urol Clin North Am. 2014 Feb;41:1-17. Review.

References on microsurgical reconstruction Vasovasostomy: 1. Belker, A.M.: Urologic microsurgery - current perspectives. I. Vasovasostomy. Urology 14:325, 1979. 2. Belker, A.M.: Vasovasostomy. In Resnick, M.I. (Ed.), Current Trends in Urology, Vol. I., Williams & Wilkins, Baltimore, pp. 20-41, 1981. 3. Belker, A.M.: Vasovasostomy and vasoepididymostomy. AUA Update Series, Volume I, Lesson 2, AUA, Inc. Office of Education, Houston, TX, 1981. 4. Sharlip, I.D.: The significance of intravasal azoospermia during vasovasostomy: answer to a surgical dilemma. Fertil. Steril. 38:496, 1982. 5. Kelami, A.: "Infrapubic" approach in operative andrology. Urology 12:580, 1978. 6. Belker, A.M.: Infrapubic incision for specific vasectomy reversal situations. Urology 32:413, 1988. 7. Kaye, K.W., et al: Infrapubic incision in operative andrology. Urology 21:524, 1983. 8. Belker, A.M., et al: Microsurgical two-layer vasovasostomy: laboratory use of vasectomized segments. Fertil. Steril. 29:48, 1978. 9. Silber, S.J.: Vasectomy and its microsurgical reversal. Urol. Clin. North Am. 5:573, 1978. 10. Belker, A.M.: Microsurgical two-layer vasovasostomy: simplified technique using hinged folding- approximating clamp. Urology 16:376, 1980. 11. Belker, A.M.: Technical aids for vasovasostomy. Urology 20:635, 1982. 12. Belker, A.M., et al: Results of 1,469 microsurgical vasectomy reversals by The Vasovasostomy Study Group. J. Urol. 145:505, 1991. Vasoepididymostomy: 13. Silber, S.J.: Microscopic vasoepididymostomy: specific microanastomosis to the epididymal tubule. Fertil. Steril. 30:565, 1978. 14. Fogdestam, I., and Fall, M.: Microsurgical end-to-end and end-to-side epididymovasostomy to correct occlusive azoospermia. Scand. J. Plast. Reconstr. Surg. 17:137, 1983. 15. Fogdestam, I., et al: Microsurgical epididymovasostomy in the treatment of occlusive azoospermia. Fertil. Steril 46:925, 1986. 16. Thomas, A.J., Jr.: Vasoepididymostomy. Urol. Clin. N.A. 14:527, 1987. 17. Berger, R.E.: Triangulation end to side vasoepididymostomy. J. Urol. 159:1951, 1998. 18. Marmar, J.: Modified vasoepididymostomy with simultaneous double needle placement, tubulotomy and tubular invagination. J. Urol. 163:483, 2000. 19. Niederberger, C. and Ross, L.S.: Microsurgical epididymovasostomy: predictors of success. J. Urol. 149:1364, 1993. 20. Chan, P. T.K., et al: Microsurgical vasoepididymostomy: a prospective randomized study of 3 intussusception techniques in rats. J. Urol. 169:1924, 2003. 21. Chan, Lee, Li, Libman and Goldstein: AUA 2008 Annual Meeting Abstract #1727. (Longitudinal intussusceptions vasoepididymostomy resykts in human patients) 22. Schiff, J., et al: Outcome and late failures compared in 4 techniques of microsurgical vasoepididymostomy in 153 consecutive men. J. Urol. 174:651, 2005. Intraoperative sperm harvesting and cryopreservation: 23. Belker, A.M. and Bergamini, D.A.: The feasibility of cryopreservation of sperm harvested intraoperatively during vasectomy reversals. J. Urol. 157:1292, 1997. 24. Boyle, K.E., et al: Sperm harvesting and cryopreservation during vasectomy reversal is not cost effective. Fertil. Steril. 85:961, 2006. Microsurgical techniques: 25. Acland, R.D.: Instrumentation for microsurgery. Orthop. Clin. N. A. 8:281, 1977. 26. Acland, R.D.: Notes on the handling of ultrafine suture material. Surgery 77:507, 1975. 27. 0'Brien, B. McC.: Microvascular Reconstructive Surgery, Churchill Livingstone, Edinburgh, 1977. 28. Horenz, P.: The operating microscope. I. Optical principles, illumination systems and support systems. J. Microsurg. 1:364, 1980. 29. Horenz, P.: The operating microscope. II. Individual parts, handling, assembling, focusing and balancing. J. Microsurg. 1:419, 1980. 30. Horenz, P.: The operating microscope. III. Accessories. J. Microsurg. 2:22, 1980. 31. Horenz, P.: The operating microscope. IV. Documentation. J. Microsurg. 2:126, 1980. 14 32. Horenz, P.: The operating microscope. V. Maintenance and cleaning. J. Microsurg. 2:179, 1981. 33. Belker, A.M.: Microsurgery for the urologist. AUA Update Series, Volume 3, Lesson 1. AUA Inc. Office of Education, Houston, TX, 1983 34. Teitz, B.: Care and handling of microsurgical instruments. Microsurg. 15:155, 1994. 35. Belker, A. M. and Bennett, A. H.: Applications of microsurgery in urology. Surg. Clin. N. A. 68:1157, 1988. 36. Belker, A.M.: Principles of microsurgery. Urol. Clin. N.A. 21:487, 1994. 37. Belker, A.M.: Principles and techniques of microsurgery. In Thomas, A.J., Jr. and Nagler, H.M. (Eds): Atlas of Surgical Management of Male Infertility, Chapter 6, pp. 43-54, New York, Igaku- Shoin Medical Publishers, Inc. 1995.

Comparative expenses of microsurgical reconstruction versus sperm retrieval with IVF/ICSI: 38. Pavlovich, C.P. and Schlegel, P.N.: Fertility options after vasectomy: a cost-effectiveness analysis. Fertil. Steril. 67:133, 1997. 39. Kolettis, P.N. and Thomas, A.J., Jr.: Vasoepididymostomy for vasectomy reversal: a critical assessment in the era of intracytoplasmic sperm injection. J. Urol. 158:467, 1997.

Sperm retrieval/genetics references

1. Palermo GD, Schlegel PN, Sills ES, Veeck LL, Zaninovic N, Menendez S, Rosenwaks Z. Births following testicular sperm used with intracytoplasmic sperm injection in non-mosaic Klinefelter's syndrome. N Engl J Med 338:588-560, 1998. 2. Schlegel PN. Testicular sperm extraction: Microdissection improves sperm yield with minimal tissue excision. Hum Reprod, 14:131-135, 1999. 3. Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection: Preoperative histology can predict success of sperm retrieval. J Urol 161:112-116, 1999. 4. Janzen N, Goldstein M, Schlegel PN, Hariprashad J, Palermo GD, Rosenwaks Z. Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and ICSI for unreconstructable azoospermia. Fertil Steril 74:696-701, 2000. 5. Raman J, Schlegel PN. Aromatase inhibitors for male infertility. J Urol 167:624-629, 2002. 6. Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod 18:1660-5, 2003. 7. Raman J, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection is successful for the treatment of nonobstructive azoospermia associated with cryptorchidism. J Urol. 170:1287-1290, 2003. 8. Schlegel PN, Kaufmann J. Role of varicocelelectomy in men with non-obstructive azoospermia. Fertil Steril. 81:1585-8, 2004. 9. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology. 2005 Jun;65(6):1190-4. 10. Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval.J Urol. 2007 Apr;177(4):1447-9. 11. Hung AJ, King P, Schlegel PN. Uniform testicular maturation arrest: a unique subset of men with nonobstructive azoospermia. J Urol. 2007 Aug;178(2):608-12; discussion 612. 12. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 89:823-831, 2008; [Epub Jul 17, 2007] 13. Ramasamy R, Schlegel PN. Effect of FSH on sperm retrieval rates in men with non-obstructive azoospermia. Fertil Steril, 92:590-3, 2009. 14. Schlegel PN. Non-obstructive azoospermia: A novel surgical technique and results. Semin Reprod Med. 2009 Mar;27(2):165-70. Epub 2009 Feb 26. Review. 15. Carrell DT, Wilcox AL, Lowy L, Peterson CM, Jones KP, Erickson L, Campbell B, Branch DW, Hatasaka HH. Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss. Obstet Gynecol. 2003 Jun;101(6):1229-35. 16. Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, Campbell B. Sperm DNA fragmenatation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl. 2003;49:49-55. 17. Dabaja AA, Schlegel PN. Microdissection testicular sperm extraction: an update. Asian J Androl. 2013 Jan;15(1):35-9. 18. Schlegel PN. Aromatase inhibitors for male infertility. Fertil Steril. 2012;98:1359-62 19. Hsiao W, Stahl PJ, Osterberg EC, Nejat E, Palermo GD, Rosenwaks Z, Schlegel PN. 15 20. Successful treatment of postchemotherapy azoospermia with microsurgical testicular sperm extraction: the Weill Cornell experience. J Clin Oncol. 2011;29:1607-11. 21. Ramasamy R, Fisher ES, Ricci JA, Leung RA, Schlegel PN. Duration of microdissection testicular sperm extraction procedures: relationship to sperm retrieval success. J Urol. 2011;185:1394-7 22. Schlegel PN. Aromatase inhibitors for male infertility. Fertil Steril. 2012 Dec; 98(6): 1359-62. 23. Ramasamy R, Reifsnyder JE, Husseini J, Eid PA, Bryson C, Schlegel PN. Localization of sperm during microdissection testicular sperm extraction for men with nonobstructive azoospermia. J Urol. 2013 Feb;189(2):643-6 24. Berookhim BM, Schlegel PN. Azoospermia due to spermatogenic failure. Urol Clin North Am. 2014 Feb;41(1):97-113. 25. Bryson CF, Ramasamy R, Sheehan M, Palermo GD, Rosenwaks Z, Schlegel PN. Severe testicular atrophy does not affect the success of microdissection testicular sperm extraction. J.Urol. 2014 Jan;191(1):175-8. 26. Stahl PJ, Schlegel PN, Goldstein M. Sperm Retrieval and quality evaluation. Methods Mol Biol. 2014;1154:361-84.