In-vitro spermatogenesis resumption in men with maturation arrest: relationship with in-vivo blocking stage and serum FSH

Jan Tesarik1,2,6, Basak Balaban3, Aycan Isiklar3, Cengiz Alatas3, Bülent Urman3, Senai Aksoy3, Carmen Mendoza2,4 and Ermanno Greco5

1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, calle Gracia 36, 18002 Granada, Spain, 3 American Hospital of Istanbul, Istanbul, Turkey, 4 University of Granada, Granada, Spain and 5 European Hospital, Rome, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown previously that germ cells recovered from some men with maturation arrest can resume spermatogenesis in vitro and give rise to late elongated spermatids. This study relates the ability of germ cells to differentiate in vitro to the stage at which spermatogenesis is blocked in vivo and to the patient's serum FSH concentration. The presence of germ cells at different stages of spermatogenesis was assessed, before and after culture, by classical cytology, by fluorescence in-situ hybridization and by immunocytochemistry with a germline-specific marker. The proportion of cases of maturation arrest at the primary spermatocyte, secondary spermatocyte and spermatid stage in which in-vitro resumption of meiosis was achieved was 24.3% (9/37), 100% (3/3) and 51.1% (23/45) respectively. Serum FSH concentrations were higher than normal in most cases. However, lower values were measured in patients in whom in-vitro spermatogenesis was achieved compared with those in whom no progression was detected. These data show that, under the conditions of this study, germ cells from men with very high serum FSH concentrations (>20 IU/l) are less likely to resume spermatogenesis in vitro than those coming from men with only moderate increase (10–20 IU/l).

Key words: FSH/in-vitro spermatogenesis/maturation arrest/spermatid/spermatocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following the first reported human pregnancies and births resulting from transfer of embryos obtained by fertilizing oocytes with round (Tesarik et al., 1995Go, 1996Go) and elongated (Fishel et al., 1995Go, 1996Go) spermatids, the techniques of round spermatid injection (ROSI) and elongated spermatid injection (ELSI) into human oocytes, described in detail earlier (Tesarik and Mendoza, 1996Go), have been applied by different groups with highly variable success rates (Mansour et al., 1996Go; Amer et al., 1997Go; Antinori et al., 1997aGo, bGo; Araki et al., 1997Go; Vanderzwalmen et al., 1997Go; Yamanaka et al., 1997Go; Barak et al., 1998Go; Bernabeu et al., 1998Go; Kahraman et al., 1998Go; Sofikitis et al., 1998aGo; Sousa et al., 1999Go). Moreover, a full-term human pregnancy has been reported with the use of secondary spermatocytes (Sofikitis et al., 1998bGo). Two independent studies (Amer et al., 1997Go; Vanderzwalmen et al., 1997Go) have suggested that the chance of obtaining viable embryos with round spermatids is higher when these cells are recovered from men in whom previous examinations have revealed the presence of at least a small number of late elongated spermatids or spermatozoa as compared with those patients in whom spermatogenesis is invariably blocked at the round spermatid stage. The latter condition, termed complete spermiogenesis failure (Amer et al., 1997Go), may be caused by an inherent germ cell defect. However, it can also be produced by a hostile environment to which germ cells are exposed in the testis afflicted by a pathological process preventing them from realizing their otherwise normal developmental potential. If this is the case, round spermatids might be able to undergo further development under appropriate conditions in vitro although they are blocked in situ.

The recourse to in-vitro culture in an attempt to improve morphological and functional maturity of testicular germ cells has been encouraged by the pioneering work of Ian Craft's group (Craft et al., 1995Go; Zhu et al., 1997Go) who demonstrated, for the first time, that the fertilizing ability of testicular spermatozoa can be improved by additional in-vitro maturation. The recent observation that round spermatids, and even primary spermatocytes, from some patients with maturation arrest can develop in vitro up to late elongated spermatids that can be used with success for assisted reproduction (Tesarik et al., 1999aGo, bGo) indicates that, under appropriate culture conditions (Tesarik et al., 1998aGo, bGo), a subpopulation of germ cells that are blocked in vivo at meiotic and postmeiotic stages of spermatogenesis can overcome the block and undergo further differentiation. However, germ cells from some men with in-vivo maturation arrest remain blocked under the same culture conditions that allow germ cells from other men with the same diagnosis to differentiate (Tesarik et al., 1999aGo). No clinical or laboratory feature according to which the success or failure of germ cell in-vitro culture can be predicted is currently available.

In this study, the ability of germ cells from patients with maturation arrest to resume spermatogenesis in vitro was evaluated in relation to the stage at which spermatogenesis was arrested in vivo and to the serum FSH concentration.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study involved 93 men, aged between 23 and 52 years, suffering from non-obstructive azoospermia. Testicular size was normal in 55 patients and unilaterally or bilaterally reduced in 38 patients. Fourteen of the patients had undergone a previous assisted reproduction attempt during which no spermatozoa were found in testicular biopsy samples and the treatment cycle was cancelled.

Samples of testicular tissue were obtained by open testicular biopsy from multiple sites of both testes. Pieces of tissue were plunged immediately in modified Earle's balanced salt solution (ME) and minced with the use of two sterile microscope slides followed by repeated vigorous aspiration into a tuberculin syringe. ME was prepared by supplementing Earle's balanced salt solution (Sigma, St. Louis, MO, USA) with penicillin G (75 mg/l), pyruvic acid (11 mg/l), human serum albumin (10 mg/ml), and HEPES (5 ml of 1 mol/l HEPES solution added to 995 ml of medium). All the additives were from Sigma except for human serum albumin which was from Irvine Scientific (Irvine, CA, USA). All tissue samples from the same patient were mixed together, and large fibrotic pieces not containing seminiferous tubules were removed with forceps.

In the first series of cases (n = 8), each testicular biopsy sample was mechanically disintegrated, homogenized and divided into three parts. The first part was fixed immediately for the cytological and histological analysis (see below). The second part was cultured for 24 h at 30°C in ME medium supplemented with 25 mIU/ml recombinant human FSH (Puregon; Organon, Oss, The Netherlands). The choice of the culture temperature was based on preliminary experiments with testicular biopsy samples from men with non-obstructive azoospermia (n = 5), showing a significantly better germ cell survival at 30°C as compared with 34°C or 37°C, and on the previously published data on in-vitro differentiation of germ cells from men with normal spermatogenesis (Tesarik et al., 1998aGo, bGo). The cell concentration in the culture tubes was approximately 1x106/ml. The third part was cultured under the same conditions but for the omission of FSH in culture medium to serve as a control. Each of the three parts was finally further divided into two equal specimens. One of them was treated for 1 h, at 37°C, with collagenase I (1000 units/ml) and elastase (10 units/ml) (Sigma), washed in phosphate-buffered saline (PBS), smeared on slides, fixed with 5% glutaraldehyde in 0.05 mol/l cacodylate buffer (pH 7.4) and analysed by immunocytochemistry with 4D4 monoclonal antibody against the germline marker proacrosin (Tesarik et al., 1998aGo). Individual stages of germ cell development, including postzygotene primary spermatocytes, secondary spermatocytes, and normal (Sa, Sb1, Sb2, Sc and Sd) and abnormal (Saf, Sbp and Scp) spermatid forms were distinguished by using the previously described criteria (Tesarik et al., 1998aGo, bGo). The other specimen was fixed with Bouin's solution without any enzymatic treatment, embedded in paraffin, serially sectioned and stained with haematoxylin and eosin for histological evaluation. Primary and secondary spermatocytes and round (Sa) spermatids were distinguished according to the standard anatomopathological criteria based on the cell size, nuclear staining and the cell position within the seminiferous tubule. For microscopic evaluation, preparations were blinded so that the person performing the evaluation was not aware of the nature of individual preparations.

In the second series of cases (n = 25), the control incubations without FSH and the examinations of fresh and cultured tissues in histological preparations were omitted. All the other procedures, including tissue homogenization, in-vitro culture and preparation of smears for cytological analyses were the same as in the first series. In addition to the immunocytochemical visualization of the germline marker proacrosin (see above), at least two smear preparations from each patient and from each treatment group were processed, respectively, for Papanicolaou staining (World Health Organization, 1992Go), two-colour fluorescence in-situ hybridization (FISH) with probes for chromosomes 15 and 16 (D15Z1 and D16Z1 respectively) and one-colour FISH (probe D15Z1) combined with proacrosin immunocytochemistry (Tesarik et al., 1998bGo). The use of FISH facilitated the distinction of meiotic stages of spermatogenesis, particularly those of secondary spermatocytes whose size and immunostaining pattern with anti-proacrosin monoclonal antibodies is similar to that of round spermatids.

In the third series of cases (n = 60), FISH was omitted, and the culture medium supplemented with 25 IU/l pure FSH (Serono, Rome, Italy) instead of recombinant FSH. All the other methods were the same in the three series.

Other cases in which late elongated spermatids or spermatozoa were unexpectedly found in fresh samples (n = 7) were excluded from further evaluation.

A blood sample was taken from each patient during the first consultation and serum FSH concentration was determined by immunoenzymology using a commercial kit (FSH-ACS:180; Chiron, Cergy-Pontoise, France; sensitivity threshold, 0.3 IU/l).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the first series of cases, the method of germ cell identification using smears prepared from enzyme-digested tissues proved more sensitive as compared with histological analysis of serial sections (Table IGo). This was already true for freshly processed biopsied material, where the examination of serial sections never revealed the presence of more advanced germ cells as compared with the smears prepared from the same samples; on the contrary, more advanced cells were detected in the smear preparation in one patient (no. 2). However, the advantage of the smear preparations was even more evident in cultured samples in which the smear technique revealed more advanced stages than the serial section technique on six different occasions, and the opposite never occurred (Table IGo). The morphological abnormalities detected in the elongating spermatids from cultured samples are typical of in-vitro matured spermatids (Tesarik et al., 1998aGo, bGo), and such spermatids can be used with success for micromanipulation-assisted fertilization (Tesarik et al., 1999aGo, bGo). The results of this series of experiments were concordant with previously published data (Tesarik et al., 1998aGo, bGo) showing that medium supplementation with FSH is beneficial for in-vitro differentiation of germ cells from men with normal spermatogenesis. Based on the present and the previous findings, the control incubations without FSH and the time- and labour-consuming serial sectioning method were omitted in the other two experimental series. As compared with undigested samples, the inclusion of the enzymatic digestion step (collagenase I and elastase) slightly increased the number of germ cells detected, but it did not lead to the identification of more advanced developmental stages.


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Table I. Cytological (smears) and histological (sections) comparison of the most advanced stages of spermatogenesis detected in fresh testicular biopsy samples (within the first hour after recovery) and after their subsequent in-vitro culture (24 h after recovery) in unsupplemented medium and in medium supplemented with 25 IU/l recombinant FSH
 
In the second series of cases, cultured samples that had not been exposed to enzymatic digestion showed the presence of more advanced stages of spermatogenesis as compared with freshly recovered samples in 14 out of 25 cases (56%). The most advanced stages of spermatogenesis detected in freshly recovered and in-vitro cultured samples from each patient are shown in Table IIGo. To increase stringency, the most advanced stage in a fresh sample was considered to be that at which at least one apparently normal germ cell was found, whereas the identification of at least 10 cells was required for the most advanced stage after in-vitro culture. In two cases, in-vitro spermatogenesis progressed up to the late Sd stage at which in-vitro-developed elongated spermatids produced tiny, but clearly visible, movements of the flagellum. In two other cases spermatids did attain the Sd stage, but their flagella remained immotile (Table IIGo). Identical results were obtained with all the methods of germ cell identification used in this series of cases.


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Table II. Comparison of the most advanced stages of spermatogenesis detected in fresh testicular biopsy samples (within the first hour after recovery) and after their subsequent in-vitro culture (24 h after recovery) with 25 IU/l recombinant FSH
 
Table IIGo also shows serum FSH concentrations of the patients from which the samples were obtained. The difference between the mean (± SD) values of serum FSH measured in patients whose germ cells showed in-vitro development (14.4 ± 3.9 IU/l) and in those whose germ cells did not (21.5 ± 6.7 IU/l) was statistically significant (P < 0.01; Student's t-test).

In the third series of cases, patients were subdivided into two subgroups according to the stage at which their spermatogenesis was blocked in vivo. From 31 patients with maturation arrest at the primary spermatocyte stage, spermatogenesis was resumed in vitro in seven cases (Table IIIGo). As in the second series, FSH concentrations in these patients (15.9 ± 4.1 IU/l) were significantly lower (P < 0.01) as compared with the remaining patients of this group (27.7 ± 4.3 IU/l).


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Table III. Results of in-vitro culture and serum FSH concentrations in men with in-vivo maturation arrest at the primary spermatocyte stage
 
From 29 patients with maturation arrest at the round spermatid stage, spermiogenesis was resumed in vitro in 14 cases and progressed up to the late elongated spermatid stage (Sd) in eight of them (Table IVGo). As in the above group, FSH concentrations were significantly lower (P < 0.01) in those patients whose spermatids resumed spermiogenesis (13.5 ± 4.3 IU/l) than in those whose spermatids did not (24.4 ± 5.4 IU/l).


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Table IV. Results of in-vitro culture and serum FSH concentrations in men with in-vivo maturation arrest at the round spermatid stage
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Based on experimental studies carried out mainly in the rat, mammalian spermatogenesis in vitro is considered to be extremely difficult to achieve, and virtually impossible without the use of long-term culture systems employing polarized Sertoli-cell monolayers (reviewed in Kierszenbaum, 1994). It is thus surprising that we could obtain in-vitro progression of spermatogenesis by culturing testicular cells for only 1 day from tissue sampling. This is even more interesting with regard to the fact that a relatively simple culture system was used. The addition of FSH to culture medium has previously been shown to accelerate meiotic reduction, nuclear condensation and spermatid elongation in cultured testicular biopsy samples from patients with obstructive azoospermia (Tesarik et al., 1998aGo, bGo). It appears therefore that the requirements for germ cell in-vitro differentiation are different in rodents and in humans. It is possible that the persistence of intact portions of seminiferous tubules is needed to produce a spermatogenesis-promoting environment. In fact, no progression of spermatogenesis has previously been observed in sample aliquots in which the seminiferous tubules were completely disintegrated by enzymatic treatment (Tesarik et al., 1998aGo). However, these observations can also be explained by cell damage due to the action of the enzymes used for tissue disintegration.

Interestingly, early studies reporting in-vitro spermatogenesis in the rat (Parvinen et al., 1983Go; Toppari and Parvinen, 1985Go) also used the culture of intact segments of seminiferous tubules. In contrast, the culture of isolated rat round spermatids on Sertoli cell monolayers did not lead to spermatid elongation even though the spermatid–Sertoli cell contact was re-established (Cameron and Muffly, 1991Go). It is thus possible that the persistence of the initial Sertoli cell–germ cell contacts stimulates the production by Sertoli cells of humoral factors that can positively influence even those germ cells that are outside, but in the proximity of, the intact seminiferous tubule segments.

From the physiological point of view, the rapidity of spermatogenic events under the in-vitro conditions clearly represents an unusual situation. A similar rapid progression has previously been observed in testicular biopsy samples from men with obstructive azoospermia and normal spermatogenesis (Tesarik et al., 1998aGo, bGo). This developmental acceleration may be explained by the action of presumptive hormone or growth factor antagonists that are active in situ but are diluted or inactivated after tissue removal from its natural context. Differences in the synthesis and activity of hormone and growth factor receptors may also exist between the in-vivo and in-vitro systems. For example, it has been shown that multiple promoter elements contribute to the activity of the FSH receptor gene in Sertoli cells, and the requirements for their activation are not the same in vivo and in vitro (Heckert et al., 1998Go). Another hypothesis, suggesting that the suppression of a developmental checkpoint controlling the synchronization of individual events occurring during spermiogenesis may be involved in the in-vitro acceleration of germ cell differentiation, has been proposed (Tesarik et al., 1999bGo). Further study is needed to elucidate the molecular events underlying these empirical findings.

The observation that immature germ cells whose development is blocked in vivo can eventually resume spermatogenesis in vitro is intriguing, and is complemented by the recent demonstration showing that some of the cultured germ cells can give rise to gametes with a full reproductive capacity (Tesarik et al., 1999aGo, bGo). These observations raise a question about the mechanism of this unexpected improvement. In this study we show that the serum FSH concentration of those patients whose germ cells are capable of resuming spermatogenesis in vitro is significantly lower compared with patients whose germ cells remain blocked. In particular, FSH concentrations exceeding 20 IU/l predict poor prognosis, whereas concentrations between 10 and 20 IU/l are often associated with good in-vitro differentiation potential of germ cells. In-vitro differentiation of germ cells from men with normal spermatogenesis requires the supplementation of culture medium with relatively high (>=25 IU/l) concentrations of FSH (Tesarik et al., 1998bGo). It is possible that the concentration of FSH used in this study (25 IU/l), though effective in samples from men with normal spermatogenesis in whom serum FSH concentrations are low, is insufficient for men with high serum FSH concentrations which may lead to desensitization of FSH receptors on Sertoli cells. It remains to be determined whether the refractoriness of germ cells from men with elevated serum FSH concentrations can be overcome by increasing the concentration of FSH in culture medium.

It is also possible that, as compared with this study in which testicular biopsy samples were cultured for 24 h only, better results might have been obtained if a longer incubation time had been used, or if the FSH effect had been potentiated by simultaneous addition of testosterone to the culture medium as described previously (Tesarik et al., 1998bGo). The first human birth after transmeiotic in-vitro differentiation of primary spermatocytes into elongated spermatids has been reported recently after culture of testicular biopsy samples in medium supplemented with both FSH and testosterone for 48 h (Tesarik et al., 1999aGo). It is thus possible that improved culture conditions would lead to in-vitro differentiation of germ cells, even in some of those cases in which no progression of spermatogenesis was observed under the conditions used in this study. This possibility is currently being evaluated.


    Notes
 
6 To whom correspondence should be addressed.E-mail: cmendoza{at}goliaf.ugr.es Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on December 22, 1999; accepted on February 23, 2000.