To determine if a semen sample is truly azoospermic, centrifugation of the semen sample with meticulous microscopic examination of the pellet is necessary. Although this might seem obvious, Ron-El et al. (1997) reported that sperm was found on extended sperm analysis of a centrifuged semen specimen in up to 35% of men who were thought to have non-obstructive azoospermia. In addition, we have found that up to 10-20% of men who have inadequate sperm on preoperative semen analysis will actually have sperm usable for ICSI on the day of oocyte retrieval. Therefore, we always repeat a semen analysis on the day of planned sperm retrieval for men with non-obstructive azoospermia.
For all patients with azoospermia, a complete history and physical examination is necessary to identify potentially correctable causes of male factor infertility. Typically, the man with non-obstructive azoospermia will have small testes (< 15 cc) with a flat epididymis. Some men may have a history of cryptorchidism. Hormonal evaluation of a man with non-obstructive azoospermia (NOA) will typically demonstrate an elevated serum FSH, with normal or nearly normal testosterone and estradiol levels. Prior to further intervention, we will usually treat any correctable abnormalities that are found on evaluation of a man with NOA, including surgical repair of large varicoceles, correction of hormonal abnormalities, and avoidance of gonadal toxins for at least three months prior to attempted TESE. Orchiopexy and varicocele repair are usually only considered if female age is less than 38, because with advanced female age the window of opportunity to achieve pregnancy is limited and these procedures benefit only a small percentage of men and require six months of recovery prior to attempted sperm retrieval.
However, with advanced female age (>38) the window of opportunity to achieve a pregnancy is limited, making orchiopexy and varicocele repair not worthwhile because they benefit only a small percent of men and require six months of recovery prior to attempted TESE.
Scrotal ultrasound
The ASRM/AUA Practice Guidelines do not recommend routine application of scrotal ultrasound for evaluation of the infertile male, although ESHRE guidelines do support routine ultrasound examination. Scrotal ultrasound should be strongly considered for patients with a history of cryptorchidism or prior germ cell tumors, and for men who have any unexplained abnormalities on testicular examination. In addition, it should be considered f body habitus limits physical examination of the scrotum or if the findings on physical examination are equivocal. Routine use of a scrotal ultrasound to screen for subclinical varicoceles is not recommended, as repair of very small varicoceles has not been shown to be effective in improving semen parameters or fertility. Ultrasound may also be of value in following patients after testicular biopsy, since intratesticular hematomas or scar tissue formation commonly occur and affect the timing for subsequent sperm retrieval procedures.
Genetic Abnormalities and Testing
In this section, I will emphasize common genetic disorders associated with spermatogenic failure, including non-obstructive azoospermia. These abnormalities include both chromosomal abnormalities, detectable with routine karyotype testing, and Y chromosome microdeletions, so called "AZF defects." Other rare genetic causes of male infertility are nicely reviewed in Mak and Jarvi: J Urology 156:1245-57, 1996. For men with severe male factor infertility, including sperm concentrations less than 10 x 106/cc and non-obstructive azoospermia, karyotype evaluation and Y chromosome microdeletion analysis is recommended before treatment with assisted reproduction.
Evaluation of a sequential series of 170 men with non-obstructive azoospermia who were candidates for TESE at Weill Cornell revealed that 17% of these men had definable genetic defects (Y chromosome microdeletions or karyotype abnormalities; Rucker et al., 1998.) We have found that the knowledge of having a genetic defect leads many men to pursue options other than TESE-ICSI and that, regardless of treatment choice, the majority of men find it reassuring to know the cause of their infertility.
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Karyotype evaluation
The most common karyotypic abnormality in men with severe male factor infertility is Klinefelter syndrome, affecting up to 7-13% of azoospermic men. Almost all men with the "pure, classic form" (47,XXY) of Klinefelter will be azoospermic, whereas limited sperm production is commonly found in men with a mosaic pattern of Klinefelter syndrome. It was previously felt that only spermatogonia with a 46,XY complement could produce spermatozoa (Martini et al., Hum Reprod 11:1638, 1996); however, recent observations indicate that a significant proportion of 24,XY spermatozoa are present in the testes of men with Klinefelter (Cozzi et al., Hum Genet 93:32, 1994). General teaching has suggested that men with Klinefelter syndrome can be readily identified by their typical physical appearance of tall stature, gynecomastia and small, firm testes. Again, however, recent observations contradict this statement. I have detected two men in my practice who were normally masculinized, between 5'6" and 5'10" in height, but had non-mosaic Klinefelter syndrome. Observations reported by Oates et al. (J Urol 155:476A, abstract 660; 1996) at the AUA Annual meeting also confirm that some men with chromosomal abnormalities will have an otherwise normal phenotypic appearance except for their infertility. Most men with Klinefelter syndrome have sperm retrievable with testicular sperm extraction (TESE) and can have children with ICSI. We have attempted treatment of over 15 men with this condition and eight children have been born to these couples. Other karyotypic abnormalities identified include Robertsonian translocations, chromosomal inversions and sex chromosome abnormalities.
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Y chromosome (AZF) microdeletions
Several genes have been identified in the distal portion of the long arm of Y (Yq) that are frequently deleted in men with non-obstructive azoospermia. The best described gene has multiple copies, and is referred to as DAZ (deleted in azoospermia.) Deletions involving DAZ were identified in 13% (12/89) of azoospermic men screened by Reijo et al. in 1995. In addition, Reijo et al’s evaluation of men with severe azoospermia revealed that 6% (2/35) had DAZ deletions. Other investigators have found longer deletions of the Y chromosome associated with male infertility. Vogt et al. have suggested that three relatively discrete regions of Yq, AZFa, AZFb, and AZFc, are deleted in severely infertile men and that the deleted region determines the chance of sperm production. Reijo et al. have shown similar deletions in men with different levels of sperm production, suggesting that other genetic or environmental factors may affect the phenotype of sperm production in men with Y chromosome microdeletions.
Several investigators have found that 3-18% of men with severe sperm production abnormalities, including azoospermia, have Y chromosome deletions. However the literature is difficult to evaluate because the data was generated in multiple laboratories, each looking at different patient populations and examining different regions and sites on Yq. For example, some investigators considered a microdeletion present when only a single ?(STS) was absent, while others considered a microdeletion present only if sequential sites on the Y chromosome failed to amplify with PCR-based analysis. Nevertheless, all investigators consistently found Y microdeletions in a measurable proportion of severely subfertile men, with no detectable deletions in normal fertile men, nor in the fathers or brothers of men with Y microdeletions.
Y chromosome deletions affecting fertility usually involve deletion of one or more entire AZFa, AZFb, or AZFc regions. An additional region of the Y chromosome referred to as AZFd has been described, however, it has been determined that AZFd is within AZFc, has no prognostic significance, is not associated with impaired sperm production, and therefore such deletions should not be reported, as they are clinically irrelevant. The specific region that is missing on the Y chromosome may provide prognostic significance. Approximately 50% of men with deletions involving only AZFc, have sperm present in the ejaculate. In azoospermic men with AZFc deletions, sperm production is commonly present within the testicle, and TESE is as successful as for other men with non-obstructive azoospermia. At Weill Cornell, sperm was found by TESE in 70% of men with AZFc deletions and azoospermia..
For men with deletions involving the AZFb region, the chance of having sperm in the ejaculate or finding sperm with TESE is severely decreased. Sperm was found in zero out of 23 men we evaluated with deletions involving AZFb who had a biopsy or sperm retrieval attempted with TESE (Hopps et al., 2003). Silber et al. also reported that in a series of five men with deletions involving both AZFb and AZFc no sperm was found on attempted sperm retrieval (ASRM Annual meeting, P-184, 1999). Therefore, we do not recommend that men with deletions involving the entire AZFb region undergo TESE unless use of donor sperm is planned as a back-up.
Deletions involving the entire AZFa region are also commonly associated with a Sertoli cell-only pattern on diagnostic biopsy (Kamp et al., 2001). Overall, approximately 9% of men with Sertoli cell-only pattern are estimated to have AZFa deletions. The need to discriminate between partial and complete deletions of an AZF region is reflected in the observation that at least one patient with a deletion of part of the AZFa region had germ cells on testis biopsy, however, to-date no sperm has been retrieved from men with complete deletions of AZFa or AZFb. Because the documented number of cases in the literature is limited, absolute predictive statements are not possible to make at this time. However, the prognosis is clearly different and dramatically worse for men with complete AZFa and AZFb deletions than for other patients with non-obstructive azoospermia.
Because Y chromosome abnormalities, including deletions, will be passed on to any male child who is produced after assisted reproduction, these men must have genetic counseling prior to treatment. In an important preliminary study of fathers and ICSI-derived sons, Kent-First et al. (Lancet 348:332, 1996) found that 10% (3/32) of unselected ICSI-derived boys had detectable Y chromosome microdeletions, however, only one of the three boys had a father with Y chromosome microdeletions detected on testing of his peripheral blood. These results suggest that mosaicism with germ-line deletions on the short segments of the Y chromosome frequently develop in spermatozoa of men with severe male factor infertility. Since men with these genetic defects have rarely or never fathered children naturally, it is uncertain whether any medical conditions will be present in the offspring with Y chromosome microdeletions, except for infertility. This knowledge makes genetic counseling difficult. On the other hand, common sense suggests that because the fathers are otherwise healthy and normal, the presence of a Y chromosome microdeletion does not pose a high risk for major congenital defects in potential offspring. However, the answer will not be available for many years, when the children born from this process are adults.
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Hormonal therapy
Hormonal therapy has never been demonstrated in randomized controlled trials to increase sperm production or quality in men with normal hormone levels. Therefore, I use hormone therapy only in men with demonstrated hormone abnormalities. Hormonal evaluation of a man with non-obstructive azoospermia (NOA) will typically demonstrate an elevated serum FSH, with normal or nearly normal testosterone levels. However, many men with NOA have an abnormal testosterone (ng/dL) to estradiol(pg/mL) ratio (T/E2 ratio), which is correctable with treatment using aromatase inhibitors like testolactone or anastrozole (Pavlovich et al., 2001). Whereas normal fertile men will have a T/E2 ratio of 16 + 3; men with NOA have a ratio of 7, and men with Klinefelter syndrome have a ratio of 4. The inbalance is correctable with aromatase inhibition, or delivery of exogenous testosterone, suggesting that increased aromatase activity from relative Leydig cell hyperplasia within the testicle may be the cause of this phenomenon. Correction of abnormal T/E2 ratios in men with severe oligospermia can effect dramatic improvements in sperm concentration and motility. We now routinely evaluate testosterone and estradiol levels for men with non-obstructive azoospermia or severe oligospermia. Men with low testosterone and a low T/E2 ratio are routinely treated with anastrazole, one miligram per day (Raman & Schlegel, 2002.) For men with Klinefelter syndrome, response is better using testolactone, 50-100 milligrams per day. Testosterone levels increased from 190 ng/dL to 332 ng/dL during testolactone treatment of these Klinefelter syndrome patients, with improved T/E ratios.
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Varicocele repair
The role of the varicocele in male infertility continues to be controversial. As early as 1987, the World Health Organization stated that varicoceles are associated with decreased male fertility. Numerous uncontrolled studies have suggested increased sperm count, motility and morphology after varicocele repair. However, few randomized controlled studies have documented a beneficial effect of varicocele repair on male fertility. A recent meta-analysis published in Lancet reported little beneficial effect of varicocele repair on male fertility (Evers & Collins, 2003). Unfortunately, this review included studies in which patients were treated for “subclinical varicoceles,” a practice that is not condoned by even the most ardent supporters of varicocele repair.
Of more relevant interest is the increasing practice of varicocele repair for men with non-obstructive azoospermia. One recent study found a return of sperm to the ejaculate in 43% (12/28) of men with non-obstructive azoospermia at an average follow-up of 24 months. A second study found spermatogenesis adequate to produce sperm in the ejaculate of 55% (12/22) of men with non-obstructive azoospermia. Other smaller and older studies have conflicting results. I recently reviewed a series of patients with varicoceles and non-obstructive azoospermia who were evaluated and treated at Weill Cornell. Of 31 men who underwent varicocele repair for documented non-obstructive azoospermia, 20% (7/31) had sperm on at least one postoperative semen analysis. However, only 9.6% (3/31) of men after varicocele repair had adequate motile sperm in the ejaculate for ICSI (without TESE). A history of prior varicocele repair did not affect the results of TESE, for men with non-obstructive azoospermia and varicoceles. Retrospective analysis of patients with clinical varicoceles identified before TESE shows that the rate of sperm retrieval was identical in those who had their varicoceles repaired before TESE as compared with those who did not have them repaired before TESE, 60% (41/68) and 60% (42/70) respectively (Schlegel & Kaufman, 2004.) Based on these data, varicocele repair is of limited value for men with non-obstructive azoospermia and varicoceles. In my opinion, surgical repair should probably be reserved for men who are very young and have large varicoceles, and possibly for those with testicular atrophy associated with a varicocele.
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Diagnostic biopsies in men with presumed non-obstructive azoospermia
The diagnosis of NOA can only be definitively made on testicular biopsy. Testis biopsy can also rule out the unlikely possibility of testicular intratubular germ cell neoplasia (carcinoma-in-situ) that is more common in men with unexplained unilateral testicular atrophy or with a history of cryptorchidism.
The most advanced spermatogenic pattern, as opposed to the predominant pattern, appears to affect the results of sperm retrieval. For men who had at least one area of hypospermatogenesis present on diagnostic testis biopsy, spermatozoa were retrieved in 81% (31/39) of attempts, whereas for men with maturation arrest as the most advanced pattern, spermatozoa were retrieved inonly 42% (8/19) of attempts. If the entire diagnostic biopsy had a Sertoli cell-only pattern, then sperm were retrieved in 24% (5/21) of TESE attempts. Although no finding absolutely determined sperm retrieval or negated the possibility of successful TESE, the findings of diagnostic biopsy were helpful in evaluating the chance of success with TESE (Su et al., 1999). In addition to the role of diagnostic biopsy in identifying rare cases of intratubular germ cell neoplasia (carcinoma-in-situ) and confirming the diagnosis of non-obstructive azoospermia, diagnostic biopsy helps to predict the chance that a TESE procedure will obtain sperm.
Testicular biopsy will help to determine the prognosis for sperm retrieval, but it will not provide definitive proof of whether sperm will be found with a more intensive evaluation of the testis (TESE or microdissection TESE). Therefore, I do not require a diagnostic testis biopsy prior to TESE-ICSI for non-obstructive azoospermia. A diagnostic biopsy should be performed if the etiology of azoospermia is not clear, if the risk of carcinoma-in-situ is high (rare), or if the results of biopsy will affect the couple’s choice to undergo TESE-ICSI. The reasons to determine the etiology are multiple (as previously discussed):
If a biopsy is performed, testicular tissue should be frozen for possible subsequent use. I perform diagnostic testis biopsies if a couple will not proceed to TESE-ICSI with only a 25% chance of sperm retrieval. If a uniform pattern of Sertoli cell-only is discovered, the couple has obtained useful information from the biopsy and avoided unnecessary ovarian stimulation.
