1 Division of Endocrinology and Master in Andrological Sciences, New Methodologies in Human Reproductive Medicine, Department of Internal Medicine and 2 Department of Human Anatomy University of Catania, Catania, Italy
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Abstract |
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Key words: azoospermia factor/germinal epithelium/oligoazoospermia/spermatogenesis/Y chromosome microdeletion
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Introduction |
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In an evaluation of the testicular biopsies of a cohort of oligoazoospermic patients carrying Yq microdeletions (Vogt et al., 1996), it was proposed that microdeletions were clustered in three different regions of the Yq, named AZFa, AZFb and AZFc, and that each of these related to a particular testicular histology. AZFa deletions are associated with Sertoli cell-only syndrome (SCOS), whereas deletions of the AZFb region are mostly associated with an arrest of spermatogenesis before or during meiosis. Both genotypes are generally detected in patients with azoospermia. On the other hand, the testicular pathology of men with microdeletions of the AZFc region could not be associated with a specific interruption phase of spermatogenesis. In these cases, most of the testicular tubules are depleted of germ cells (SCOS phenotype), whilst a more or less complete spermatogenesis is observed in other tubules and, therefore, AZFc deletions are compatible with the presence of spermatozoa in the ejaculate (Vogt et al., 1996
). Nevertheless, results from different studies have questioned the existence of a direct link between testicular histology and the site of Yq microdeletions (Qureshi et al., 1996
; Pryor et al., 1997
; Calogero et al., 1999
). Thus, a clear relationship between genotype and phenotype does not seem to exist (for reviews see Simoni et al., 1998; Krausz and McElreavey, 1999). The reason for this is that spermatogenesis is a complex biological process which depends on a precise, sequential and well-coordinated activation of autosomal and Yq genes whose precise role is still far from clear. However, other factors may also contribute to the lack of a genotypephenotype relationship; an important one may be germ cell regression over time, ultimately leading to complete SCOS. The decline of sperm count over time reported in Yq-microdeleted patients can be taken as indirect evidence of this phenomenon (Girardi et al., 1997
; Simoni et al., 1997
; A.E.Calogero et al., unpublished results). Here, we report the case of a patient with an AZFc microdeletion, who was severely oligozoospermic at the time of his first control, and became azoospermic one year later. Two testicular biopsies, obtained one year apart, demonstrated a tubular cellular regression from post-meiotic germ cell arrest, at the time when he was oligozoospermic, to SCOS when he had become azoospermic.
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Case report |
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In September 1998, because of his azoospermic status, the patient entered an intracytoplasmic sperm injection (ICSI) programme associated with testicular sperm recovery, but oocyte retrieval had to be cancelled, because spermatozoa could not be found even after four left testicular sperm aspirations (TESA) followed by a contralateral open testicular biopsy (testicular exploration and sperm extraction; TESE).
TESA and TESE were performed under local anaesthesia, which was achieved by infiltrating the homolateral spermatic cord in several parts with 58 ml of 7% ropivicaine hydrochloride monoidrate (Naropina, Astra, Milan, Italy). TESA was conducted as follows: an insulin needle attached to a 10 ml syringe with a 19-gauge needle was passed through the sterilized skin into the testis. Once the needle was inserted, a strong negative pressure was exerted and maintained, while the needle was moved gently up and down until a small liquid aliquot appeared within the microtubing set. The needle was then slowly withdrawn and a core of attached tissue was cut off on withdrawal from the skin surface. The content of the needle was flushed into a 100 µl volume of IVF medium (Medicult, Copenhagen, Denmark) in a Petri dish. This dish was then examined at x200 magnification under an inverted microscope in order to detect the presence of any spermatozoa.
TESE was conducted as follows: a 0.5 to 1 cm incision was made through the skin and the underlying layers. After incision of the albuginea, a gentle pressure was exerted on the testicular mass and a small portion of the extruding tissue was removed using curved scissors. The tissue was then fixed in Bouin's solution and embedded in paraffin wax according to standard procedures. Sections (56 µm thick) were cut, stained with haematoxylin and eosin, and observed at x100, x200 and x400 magnification under a light microscope. All cross-sectioned tubules present in a section of the testicular biopsy were evaluated systematically, and each was given a score from 1 to 10 following Johnsen's criteria (Johnsen, 1970). Four different sections were analysed for each biopsy.
Yq intactness was evaluated on genomic DNA obtained from peripheral leukocytes using a set of 19 sequence-tagged site (STS) primers (Primm, Milan, Italy), spanning throughout intervals 5 and 6 which comprised the AZFa, AZFb and AZFc regions (see Table I for the list of STSs used). Briefly, DNA (100200 ng) was amplified by PCR in a 25 µl final reaction volume, using: 2.5 µl of 10x buffer (Celbio, Milan, Italy), 0.5 µl of dNTP mix (10 mmol/l) (Celbio), 1 µl of each primer (50 nmol/l), and 0.5 µl of Taq DNA polymerase (2 IU/µl) (Celbio). Thermocycling consisted of 35 cycles each at 94°C for 1 min (melting), at 61°C for 1 min (annealing), and at 72°C for 1 min (extension). The programme was preceded by a 3 min denaturing phase at 94°C and followed by a final extension phase at 72°C for 7 min. Reaction products were stored at 4°C until they were loaded onto a 2% agarose gel and the gradients separated by electrophoresis in TAE 1x (Trisacetic acidEDTA) buffer at room temperature using a voltage gradient of 9 V/cm for 5060 min. An STS was recorded as absent after three amplification failures.
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Results |
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Discussion |
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Many factors, whether genetic or not, may weaken the possibility of establishing a genotypephenotype relationship. For example, an approximate estimation of the microdeletion extension in the various patients may be a relevant factor since deletions considered similar may, instead, include important sequences within their boundaries that impair gene expression even further. Concomitant non-genetic factors capable of impairing spermatogenesis may also play a relevant role in the lack of a genotypephenotype relationship. For example, if not carefully studied, varicocele, exposure to gonadotoxins, orchitis and/or other urogenital inflammatory processes may upset the phenotypic expression of Yq microdeletions by further damaging spermatogenesis.
The case of the microdeleted patient reported herein suggests that a lack of genotypephenotype correlation may also be due to a spontaneous progression of testicular lesion in the course of time. Sparse clinical evidence has suggested the occurrence of such a case in some Yq-microdeleted patients. Indeed, few microdeleted patients have been reported to have a decline in sperm output over time (Girardi et al., 1997; Simoni et al., 1997
). According to a recent study, a patient with Yq microdeletions was able to father four children from the age of 25 to the age of 38 but later, at the time of the Yq microdeletion assessment, was found to be azoospermic (Chang et al., 1999
). The case presented here fits in with these findings since, following an earlier sperm analysis, our patient proved to be cryptospermic and in less than two yearswithout the intervention of any other factors known to impair spermatogenesishe became azoospermic. This clinical finding implied that depletion of the tubular germ cell line may account for this phenomenon. The possibility of carrying out two sequential testicular biopsies one year apart gave us the opportunity of ascertaining that there was indeed a progressive tubular depletion of germ cells, ultimately leading to an almost complete germ cell aplasia. Although a single biopsy has been claimed to be unrepresentative of the whole testicular picture because of the heterogeneity of the testicular histology of patients with non-obstructive azoospermia (Johnsen, 1970
), there is some inconsistency in the literature concerning the testicular histology of such patients. Others (Silber et al., 1997
) have detected a multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a regional distribution. Moreover, biopsy samples obtained following fine-needle aspiration biopsy have been found to show a good correlation with conventional histology obtained by open biopsy (Mallidis and Baker, 1994
; Craft et al., 1997
). In our hands, TESA performed twice, 612 months apart, in the same patients undergoing ICSI resulted in the same cytological diagnosis in six out of seven cases. The fact that the majority of the cellular yield, following four TESA in different areas of the left testis of the microdeleted patient described in this article, consisted mainly of Sertoli cells strengthened the hypothesis of a worsening of the testicular histology, from the least severe picture of maturation arrest to the severest SCOS, without the apparent intervention of any cause known to affect germinal epithelium. This may support the argument whereby the same genetic alteration may show-up with a continuous spectrum of testicular phenotypes, depending on the time of observation, which contributes to the lack of a close relationship between genotype (position and length of the microdeletion) and phenotype (testicular biopsy and, consequently, sperm output). Although the case described herein seems to warrant such a conclusion, it is possible that this finding may not be a common feature of AZF-microdeleted patients; further investigations are therefore needed to support this hypothesis.
To our knowledge, this is the first case of spontaneous germinal cell epithelium regression in a patient with a Y chromosome microdeletion, which may in part account for the lack of a genotypephenotype correlation observed in these patients.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 15, 2001; accepted on May 14, 2001.