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Spermatid count as a predictor of response to FSH therapy
Reproductive BioMedicine Online (2014) xxx, xxx– xxx
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ARTICLE
Spermatid count as a predictor of response to FSH therapy Andrea Garolla a, Riccardo Selice a, Bruno Engl b, Alessandro Bertoldo a, Massimo Menegazzo a, Livio Finos c, Andrea Lenzi d, Carlo Foresta a,* a Department of Molecular Medicine, Section of Clinical Pathology and Unit for Human Reproduction Pathology, University of Padova, Italy; b Obstetrics and Gynaecology Unit, Brunico, Italy; c Department of Statistical Sciences, University of Padova, Italy; d Department of Experimental Medicine, Section of Medical Physiopathology and Endocrinology, University of Rome ‘La Sapienza’, Italy
* Corresponding author. E-mail address: [email protected] (C Foresta). Carlo Foresta is full Professor of Endocrinology and Director of Human Reproduction Pathology Unit at the University of Padova, Italy. During the last 30 years his research activities included different aspects related to male reproduction and spermatogenesis. Current research and clinical interests are in the genetics of male infertility, cryptorchidism and testicular tumours, endocrine regulation of spermatogenesis, sexual transmittable diseases and sexual disorders. He is author of more than 250 papers in International peer-reviewed journals, and invited speaker to many international and national scientific congresses. He is Past President of the Italian Society of Andrology and Sexual Medicine and member of the executive board of many scientific societies.
Abstract This study evaluated the predictive power of spermatid count and cytology for assisted reproduction outcome after FSH
therapy. A total of 174 men with severe oligozoospermia and normal plasma FSH concentration underwent semen analysis including spermatid count, TUNEL test, FISH analysis for sperm aneuploidies and testicular fine-needle aspiration cytology. Ninety-two men with hypospermatogenesis received FSH therapy for 3 months and 82 patients with maturative disturbance or partial obstruction served as controls. Semen was analysed at baseline, after FSH therapy and after 3- and 9-month follow up, and pregnancies were recorded. Subjects not reaching pregnancy at 3-month follow up were recommended assisted reproduction treatment. Spermatid count was related to testicular cytology: spermatid concentrations <0.01, 0.01–0.3 and >0.3 · 106/ml were predictive of partial obstruction, hypospermatogenesis and maturative disturbance. FSH therapy patients showed increases in sperm number and motility (both P < 0.001), allowing some couples to undergo intrauterine insemination instead of IVF. Cumulative pregnancy rate after 12 months was higher with FSH therapy (44.6%) than without (22.0%; P = 0.002). FSH therapy improved pregnancy rate and sometimes allowed less invasive assisted reproduction treatment in well-selected patients. Spermatid count could represent a new parameter to predict response to FSH therapy. RBMOnline ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Assisted reproduction outcome, FSH therapy, male infertility, pregnancy rate, severe oligozoospermia, spermatid count
http://dx.doi.org/10.1016/j.rbmo.2014.02.014 1472-6483/ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
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Introduction Normal functioning of the gonadotrophic axis, in particular normal FSH secretion, plays crucial roles in spermatogenesis (Matsumoto, 1989; Sharpe, 1989), and several experimental and clinical studies have demonstrated the importance of FSH in regulating a normal quantitative spermatogenic process in animals and humans (Marshall et al., 1995; McLachlan et al., 1995; Moudgal et al., 1997). It has previously been demonstrated that FSH administration is able to increase sperm concentration, spermatogonal population and pregnancy rate in oligozoospermic subjects with normal plasma concentrations of gonadotrophins (Acosta et al., 1992; Bartoov et al., 1994; Glander and Kratzsch, 1997; Merino et al., 1996; Strehler et al., 1997). In addition, some authors reported that a significant improvement of sperm quality was evident after therapy. FSH therapy is useful in reducing the apoptotic process of spermatozoa and improving the qualitative properties of the axoneme, chromatin and acrosome (Baccetti et al., 1997). Recently Colacurci et al. (2012) showed that the degree of sperm DNA fragmentation significantly decreases after 90 days of FSH therapy. However, reducing fragmentation cannot guarantee fertilization and embryo development success systematically. Some authors reported that genetic, in particular epigenetic causes, can lead to abnormal spermatogenesis, with potential implications for sperm quality, fertilization and embryo development (Jenkins and Carrell, 2012; Lalancette et al., 2009). In particular, Montjean et al. (2013) evaluated methylation profiles in mature sperm DNA from normozoospermic and oligozoospermic men by observing the presence of methylation changes in spermatozoa of the latter group. Another potential mechanism of DNA damage that could be able to reduce the capability of spermatozoa to fertilize is related to DNA denaturation (Evenson and Wixon, 2008). It has been postulated that defective chromatin condensation during spermatogenesis could lead to spermatozoa unable to mature completely during spermiogenesis and therefore unable to repair DNA breaks during chromatin rearrangement occurring when histones are replaced by protamines (O’Brien and Zini, 2005; Zini and Libman, 2006). A positive effect on sperm DNA condensation after FSH therapy has been demonstrated in idiopathic infertile men (Kamischke et al., 1998). These findings may explain the increase in oocyte fertilization and pregnancy rate, sometimes in absence of improved classic seminal parameters, that has been observed in couples undergoing assisted reproduction treatment in which the male partner received FSH (Acosta et al., 1991, 1992; Ashkenazi et al., 1999). However, the improvement in seminal characteristics is variable and not much evident in many cases, and a proportion of patients do not respond to FSH therapy. Oligozoospermia may be sustained by various alterations of the seminiferous epithelium that could explain the failure of FSH therapy reported by other studies (Attia et al., 2007; Bartoov et al., 1994; Foresta et al., 1995). Fine-needle aspiration cytology (FNAC) is a minimally invasive, rapid and effective procedure to evaluate the tubular status in oligozoospermic patients (Adhikari, 2009). This study group
A Garolla et al. demonstrated that FSH therapy of oligozoospermic subjects with normal FSH plasma concentration, moderate hypospermatogenesis and absence of maturation disturbance is able to induce both a significant increase in sperm count (Foresta et al., 2002) and an improvement in pregnancy rate (Foresta et al., 2005). In contrast, when hypospermatogenesis is associated with maturative disturbance, FSH therapy shows no effect (Foresta et al., 2009). In the light of this consideration, selection criteria should be used to derive predictive information on the response to FSH therapy. Some polymorphisms in the FSH receptor gene (FSHR) are able to influence the sensitivity of the receptor for the hormone and the reproductive parameters both in men and women (Aittomaki et al., 1995; Behre et al., 2005; Greb et al., 2005; Tu ¨ttelmann et al., 2012). On this basis, the current study group tested the hypothesis that FSHR sensitivity might be important in the response to FSH therapy and demonstrated that this treatment induces diverse improvement in seminal parameters according to defined FSHR genotypes; only subjects with at least one serine in position 680 (homozygotic AS/AS and heterozygotic TN/AS) had an improvement of seminal parameters (Selice et al., 2011). Again, molecular studies on the FSHB gene (coding for the b-subunit of FSH) showed that a G/T single-nucleotide polymorphism located in the FSHB gene promoter ( 211 bp from the mRNA transcription start site; rs10835638) is responsible for the relative activity of endogenous FSH (Grigorova et al., 2007, 2008). Finally, research showed that oligozoospermic TT homozygotes for the FSHB 211 variant have a better response to FSH therapy in terms of sperm count and motility than patients with variants GG and GT (Ferlin et al., 2011). Although the pharmacogenomic approach to male infertility seems to represent an important tool to predict the response to FSH therapy, it is still very expensive, time consuming in clinical situations and requires more and larger studies to determine tailored FSH dosage. This prospective controlled clinical study aimed to identify a new, simple, cheap and effective parameter to predict the response to FSH therapy in infertile oligozoospermic patients with normal FSH plasma concentrations in terms of sperm parameters and fertility outcome.
Materials and methods Patients and setting This study was approved by the ethics committee of the Hospital-University of Padova (protocol number 2591P, approved 21 March 2010). Informed consent was obtained from each subject. All phases of the study were performed at the Infertility Centre of the University of Padova from January 2011 to December 2012. All men were informed about the FSH therapy protocol and in particular about the different phases of the study and the possibility of undergoing assisted reproduction treatment during this period. The inclusion criteria were age 25–45 years, a history of infertility for at least 2 years, sperm count <10 · 106/ml on at least three separate occasions according to World Health Organization guidelines (WHO, 2010) and any kind of infertility cause with exclusion of seminal tract infections and antisperm antibodies, normal plasma concentrations of
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome FSH (1–8 IU/l), LH (2–8 IU/l), testosterone (10–25 nmol/l) and exclusion of female factors such as ovulatory disorders, tubal factor and endocrine abnormalities evaluated by hormone assessment, pelvic ultrasound examination and hysterosalpingography. A total of 262 patients fulfilled the inclusion criteria and were enrolled in the study. Of these, 57 subjects dropped out before completing the study and 31 couples were subsequently excluded from the study because of concurrent illnesses. Therefore, 174 men affected by oligozoospermia completed the study. All patients underwent ultrasound scanning of the testis to evaluate testicular size and morphology before undergoing testicular aspiration.
Cytological and sperm analyses Fine-needle aspiration cytology All patients underwent analysis of testicular structure by means of bilateral fine-needle aspiration cytology (FNAC) to evaluate the tubular status related to the oligozoospermia. The methods of aspiration and cytological analysis have been described previously in detail (Foresta and Varotto, 1992; Foresta et al., 1992, 1995). Briefly, testicular aspiration is a simple procedure performed with a 23-gauge (0.6 mm) butterfly needle attached to a 20-ml syringe. The retrieved material was placed on two or more microscope slides for each testis, stained with May–Gru ¨nwald and Giemsa stains and examined under a light microscope at magnifications ·125, ·400 and ·1250, counting at least 200 spermatogenic cells (spermatogonia, primary and secondary spermatocytes, early and late spermatids and spermatozoa) per smear and the interposed Sertoli cells. The cell number is expressed as a percentage and represents the mean of three small aspirations performed at cranial, central and caudal level of each testis. Semen analysis Semen evaluations were performed in a blinded fashion with regard to therapy and by the same operators (WHO, 2010). Count of round and elongated spermatids was performed as suggested by the WHO manual for the quantification of immature spermatogenic cells (WHO, 2010). After liquefaction at room temperature for 30 min, 100 ll native semen was smeared on clean grease-free slides and air dried. Slides were first stained with May–Gru ¨nwald for 3 min and then rinsed twice in tap water. Thereafter, they were stained for 15 min with Giemsa (1:10 in distilled water) and then washed again three times in tap water. Spermatid count
3 was performed by light microscopy (Nikon Eclipse E600; Nikon Corporation, Japan), according to the formula shown in Figure 1. FISH analysis The study of sperm aneuploidy was performed using multicolour fluorescence in-situ hybridization (FISH), as described elsewhere (Foresta et al., 1998), for chromosomes X, Y, 13, 18 and 21. DNA hybridization was performed using two human satellite probe-specific mixes, one for chromosomes X, Y and 18 and one for chromosomes 13, 21 and 18 (Kreatech Diagnostics). Probes were directly labelled with fluorochrome PlatinumBright495 for chromosomes X and 13, resulting in a green signal, fluorochrome PlatinumBright550 for chromosomes Y and 21, resulting in a red signal, and PlatinumBright415 for chromosome 18, resulting in a blue signal. DNA denaturation of spermatozoa and probes, incubation, post-hybridization washing and nuclear staining were performed according to the manufacturer’s instructions. After preparation, slides were observed using a fluorescence microscope (Nikon Eclipse E600) equipped with a triple band-pass filter set (fluorescein isothiocyanate (FITC), tetrarhodamine isothiocyanate and 4(,6-diamidino-2-phenylindole)). Single spots were evaluated as reported elsewhere (Robbins et al., 1995). At least 2500 cells were scored for each patient. Data are expressed as the percentage of aneuploid cells. TUNEL test Evaluation of DNA fragmentation was performed as reported by Tesarik et al. (2004). The presence of sperm DNA strand breaks was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) by means of a In situ cell detection kit (Roche Diagnostics, Germany) with FITC-labelled dUTP. For each sample, 200 spermatozoa were evaluated through the fluorescent microscope with a ·100 oil-immersion objective. Results were expressed as the percentage of cells with fragmented DNA (green staining) versus normal cells (blue staining). As positive control, this study used a normal sample treated with 30 U/ml DNase A (Roche Diagnostics) in 50 mmol/l Tris-HCl pH 7.5 and 1 mg/ml BSA for 30 min at 37C to induce DNA strand breaks before labelling. The negative control was incubated in labelled solution without TdT.
Reproductive hormone analysis Plasma FSH and LH concentrations were measured in each subject by radioimmunoassay using 125I-labelled FSH and
Figure 1 (a) Formula used to determine spermatid concentration in semen samples (WHO, 2010). (b) Semen samples stained with May–Gru ¨nwald and Giemsa; arrows indicate spermatids between mature spermatozoa; bars = 10 lm. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
4 LH (Ares-Serono, Milan, Italy). The sensitivity of both the FSH and LH assays was 0.05 IU/l, as defined by mean ± 2SD multiple zero-sample measurements. Inter- and intraassay coefficients of variation were 2.8 and 3.7% for LH, and 2.6 and 3.6% for FSH, respectively. Testosterone plasma concentrations were determined in all subjects using a double-antibody radioimmunoassay commercial kit (Radim, Rome, Italy). The assay has a sensitivity of 0.1 nmol/l and cross-reacts minimally with other steroid hormones. All specimens were measured in duplicate in the same assay. Intra- and interassay coefficients of variation were 7.8 and 7.0%, respectively.
Study design After analysis at baseline (T0), subjects were studied after three consecutive phases, with primary outcomes being represented by both sperm parameters and clinical pregnancy rate: (i) 3 months after highly purified FSH therapy (T1); (ii) 3 months after the withdrawal of therapy, during which all subjects were followed for semen parameters and spontaneous pregnancies (T2); and (iii) 6 months after assisted reproduction treatment for those who had not reached clinical pregnancy during the two previous periods (T3). Figure 2 shows the flow diagram of the whole study.
A Garolla et al. Therapy After the initial assessment (history and physical examination, basal hormone parameters, semen analysis, spermatid concentration, ultrasound scanning of the testes, FNAC, FISH analysis and TUNEL test), patients were divided into three groups on the basis of their tubular function determined by FNAC: (group A) hypospermatogenesis (n = 92); (group B) hypospermatogenesis with maturative disturbance (n = 61); and (group C) normal spermatogenesis indicating partial obstruction of seminal tract (n = 21). Because hypospermatogenesis without maturative disturbance is considered a prerequisite for response to FSH therapy, patients in group A were allocated to the therapy group and patients in groups B and C were allocated to the control group. Before therapy, all patients in group A were offered preventive sperm banking at this clinic in case the therapy was ineffective or had an adverse effect. Therapy patients received 150 IU highly purified urofollitropin i.m. three times a week for 3 months and the controls did not receive any therapy. At the end of the therapy period, semen analysis, ultrasound scanning of the testes, FISH analysis and TUNEL test were repeated in all subjects by the same operators. Preand post-therapy evaluations were performed in a blinded fashion with regard to therapy. All spontaneous pregnancies
Figure 2 Flow diagram of the study. FNAC = fine-needle aspiration cytology; IUI = intrauterine insemination; IVF-ET = in-vitro fertilization embryo transfer. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome
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occurring during this period were recorded and eventually confirmed by measurement of plasma concentrations of human chorionic gonadotrophin and by ultrasound to check the presence of a gestational sac and a fetal heart beat.
the specific indications. After this period, new pregnancies were recorded.
Follow up To evaluate the effectiveness of FSH therapy, the 174 male-factor infertile couples were followed for an additional 3-month period. All subjects were encouraged to have sexual intercourse at least two or three times per week, particularly at midcycle. All spontaneous pregnancies occurring during these 3 months were recorded. At the end of the follow-up period, all patients were re-evaluated by semen analysis.
Results are expressed as mean ± SD. Variations in FSH plasma concentrations, bi-testicular volume and motile spermatozoa between T0 and T1 are expressed as raw differences. Comparisons of groups A, B and C were performed with the ANOVA test and comparisons of the quantitative outputs of patients and controls were performed using the t-test. To evaluate the effects of FSH therapy, the twosample t-test was computed using differences with respect to T0. When the outcomes were logical (as pregnancies), Fisher’s exact test was used. The optimal discrimination rule to predict the three groups, on the basis of spermatid count, was obtained with recursive partitioning (decision tree) and the evaluation was based on cross-validated errors. The software package R (R Core Team, 2012) with package rpart were used for statistical analyses. Probability values of <0.05 were considered statistically significant.
Assisted reproduction treatment Three months after the withdrawal of FSH therapy, all couples who had not achieved spontaneous clinical pregnancy were recommended assisted reproduction treatment. For the couples who accepted treatment, those with >5 · 106/ml motile post-wash spermatozoa were enrolled to intrauterine insemination (IUI) for three cycles or IVF for one cycle (Khalil et al., 2001; Oehninger, 2000). If the number of total motile spermatozoa was not suitable for IUI (5 · 106/ml), couples were enrolled only for IVF. Either IVF–embryo transfer or intracytoplasmic sperm injection (one cycle) were performed within 6 months, considering
Statistical analysis
Results Testicular cytology obtained by FNAC and seminal smears for spermatid count from patients in each group are shown in Figure 3.
Figure 3 (a–c) Cytological images of semen obtained by fine-needle aspiration from testicles of patients with severe oligozoospermia and (d–f) seminal smears stained with May–Gru ¨nwald and Giemsa, for patients with severe hypospermatogenesis (a, d), maturative disturbance at spermatid level (b, e) and normal spermatogenesis with partial obstruction (c, f). Arrows indicate spermatids. Bars = 10 lm. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
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A Garolla et al. Table 1 Baseline clinical, hormonal and seminal features observed in 174 oligozoospermic patients according to testicular cytology. Characteristic
Group A (n = 92)
Clinical and hormonal Age (years) Bi-testicular volume (ml) FSH (IU/l) LH (IU/l) Testosterone (nmol/l)
xxx 34.1 ± 5.9 22.8 ± 4.7a,b 5.6 ± 1.9 5.7 ± 2.3 14.1 ± 4.6 xxxx xxx 2.8 ± 1.8 5.5 ± 2.8 13.8 ± 7.6 23.7 ± 18.5c 7.4 ± 5.5 0.02 ± 0.05a,b 2.1 ± 1.0d 24.4 ± 9.5
x Seminal Semen volume (ml) Sperm concentration (· 106/ml) Sperm count (· 106) Progressive motility (%) Normal morphology (%) Spermatid concentration (· 106/ml) Aneuploidy (%) DNA fragmentation (%)
Group B (n = 61)
35.2 ± 6.0 27.3 ± 6.4 4.9 ± 1.9 5.3 ± 1.9 16.2 ± 3.5
Group C (n = 21)
32.3 ± 5.4 31.9 ± 8.6 4.4 ± 1.4 5.0 ± 2.2 15.9 ± 4.5
2.5 ± 1.1 6.1 ± 2.6 14.6 ± 7.4 27.4 ± 15.3c 8.2 ± 5.4 0.77 ± 0.67d 2.3 ± 1.2d 23.4 ± 9.3
2.1 ± 1.2 5.1 ± 2.2 12.3 ± 6.8 13.2 ± 13.6 6.2 ± 4.8 0.001 ± 0.01 1.1 ± 0.6 21.4 ± 9.9
Values are mean ± SD. Group A = hypospermatogenesis; group B = maturative disturbance; group C = partial obstruction. a Versus group B: P < 0.001. b Versus group C: P < 0.001. c Versus group C: P < 0.01. d Versus group C: P < 0.001.
Table 2 Comparison of diagnoses of hypospermatogenesis, maturative disturbance and partial obstruction by FNAC and by spermatid count, obtained by segmentation tree analysis. Diagnosis by FNAC
Group A (n = 92) Group B (n = 61) Group C (n = 21)
Diagnosis by spermatid count Hypospermatogenesis (n = 92)
Maturative disturbance (n = 61)
Partial obstruction (n = 21)
88 0 4
0 61 0
4 0 17
Values are n. Group A = hypospermatogenesis; group B = maturative disturbance; group C = partial obstruction. Hypospermatogenesis = spermatid count 0.01–0.3 · 106/ml; maturative disturbance = spermatid count 3 · 106/ml; partial obstruction = spermatid count <0.01 · 106/ml. FNAC = fine-needle aspiration cytology.
Table 1 shows the baseline clinical, hormonal and seminal characteristics observed in 174 oligozoospermic patients divided into three groups on the basis of FNAC. There were 92 subjects who showed only hypospermatogenesis (group A), 61 who showed hypospermatogenesis with maturative disturbance (44 round and 17 elongated spermatidic arrest) (group B) and 21 who showed normal spermatogenesis (group C). There were no significant differences between the three groups in terms of age, duration of infertility (3.4 ± 2.5, 3.3 ± 2.1 and 3.1 ± 2.3 years), hormonal parameters and DNA fragmentation. Patients in group A had significantly smaller bi-testicular volume (P < 0.001) than in groups B and C. Patients in groups A and B showed higher percentages of motile spermatozoa and aneuploidies than in group C, while no difference in DNA fragmentation was found. The concentration of round and elongated spematids was significantly different between the three groups
(P < 0.001). Baseline aneuploidy rate resulted significantly higher in semen form groups A and B (both P values < .0.001 versus group C).
Spermatid concentration and diagnosis of cause of oligozoospermia Using segmentation tree analysis, cross-analysing the data obtained from both the spermatid concentration and FNAC analyses allowed this study to create a rule using spermatid concentration to diagnose hypospermatogenesis, maturative disturbance and partial obstruction with an acceptable level of accuracy. Patients with spermatid concentration 0.01–0.3 · 106/ml were assigned a diagnosis of hypospermatogenesis, those with spermatid concentration 0.3 · 106/ml were assigned a diagnosis of maturative
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome disturbance and those with spermatid concentration <0.01 · 106/ml were assigned a diagnosis of partial obstruction. Table 2 shows the diagnoses of cause of oligozoospermia obtained using the spermatid concentration diagnostic rule. Among the 174 patients, correct diagnosis failed just in eight (4.6%) patients: four subjects of group A were diagnosed with partial obstruction and four patients in group C were diagnosed with hypospermatogenesis. By this method, no wrong diagnoses were reported for patients of group B.
Therapy Patients in group A (n = 92) received FSH for 3 months and patients in groups B and C (n = 82) received no FSH. FSH therapy was well tolerated in all subjects, and no significant side effects were reported. No variation in seminal volumes was observed in the therapy and control groups (Table 3). In the therapy group, there were significant improvements in sperm concentration and count after therapy, with a doubling of sperm concentration with respect to T0 and the control group (both P <0.001; Table 3). The therapy group had also a significant increase of plasma FSH (from 5.6 ± 1.9 to 10.4 ± 2.3 IU/l), bi-testicular volume (from 22.8 ± 4.7 to 23.2 ± 4.6 ml) and progressively motile spermatozoa (from 2.8 ± 2.1 to 9.6 ± 5.4 · 106/ml) between T0 and T1 (P < 0.001, P < 0.05 and P < 0.001, respectively). No significant variations were observed in other seminal parameters, such as normal morphology (from 7.4 ± 5.5 to 8.3 ± 5.2%), sperm aneuploidies (from 2.1 ± 1.0 to 2.0 ± 1.1%), DNA fragmentation (from 24.4 ± 9.5 to 24.1 ± 8.2%),
Table 3 Seminal parameters throughout the study in the therapy and control groups. Parameter
Therapy group (n = 92)
Seminal volume (ml) T0 T1 T2 T3
xxx 2.9 ± 1.8 2.9 ± 1.8 2.9 ± 1.8 2.9 ± 1.8 xxxx xxx
x Sperm concentration (· 106/ml) T0 T1 T2 T3
x Sperm count (· 106) T0 T1 T2
5.5 ± 2.8 11.7 ± 5.4a 9.9 ± 4.1a 5.9 ± 3.1 xxxx xxx 13.8 ± 7.6 28.2 ± 15.9a 24.1 ± 10.7a
Control group (n = 82)
2.6 ± 1.1 2.5 ± 1.1 2.6 ± 1.1 2.6 ± 1.0
5.5 ± 2.4 5.6 ± 3.4 5.4 ± 3.5 4.7 ± 3.8
13.9 ± 7.1 14.3 ± 9.3 13.1 ± 9.1
Values are mean ± SD. T0 = at baseline; T1 = after 3 months of FSH therapy; T2 = 3 months after the withdrawal of therapy; T3 = after 6 months of assisted reproduction treatment. a Versus T0 and controls: P < 0.001.
7 plasma LH (from 5.7 ± 2.3 to 5.2 ± 2.2 IU/l) and testosterone concentration (from 14.1 ± 4.6 to 14.3 ± 4.3 nmol/l). In the control group, no significant differences were observed after therapy (Table 3). For T0 and T1, bitesticular volume from 28.5 ± 7.3 to 28.3 ± 7.4 ml, plasma FSH from 4.9 ± 1.8 to 5.0 ± 1.7 IU/l, plasma LH from 5.2 ± 2.0 to 5.4 ± 1.9 IU/l, testosterone from 16.1 ± 3.7 to 16.0 ± 3.6 nmol/l, progressively motile spermatozoa from 2.9 ± 2.0 to 3.1 ± 1.9 · 106, normal morphology from 7.7 ± 5.3 to 8.4 ± 5.6%, sperm aneuploidies from 1.9 ± 1.2 to 2.0 ± 1.1% and DNA fragmentation from 22.9 ± 9.4 to 23.5 ± 9.1%). During the therapy period, spontaneous pregnancies were observed only in the therapy group (6/92 versus 0/82 couples, P = 0.03).
Follow up Three months after the withdrawal of FSH therapy, another sperm analysis was performed in all subjects. The therapy group showed significantly higher sperm concentration and count with respect to T0 that were not different from those observed at T1 (P < 0.001; Table 3). After the follow-up period, the therapy group showed a significantly higher clinical pregnancy rate than the control group, with 19/86 (22.1%) patients achieving spontaneous pregnancies in the treated group versus 4/82 (4.9%) in the control group (P = 0.001; Table 4).
Assisted reproduction treatment Six months after the withdrawal of FSH therapy (T3), sperm analysis did not show any significant difference in seminal parameters between therapy and control patients (Table 3). During this period, assisted reproduction treatment was recommended to the patients who had not achieved pregnancy. In the therapy group, patients accepting assisted reproduction treatment were enrolled in two treatment programmes: 28 patients to IUI (three cycles) and 21 subjects to IVF–embryo transfer (one cycle). After 6 months of treatment, seven pregnancies were achieved in patients assigned to IUI (7/28, 25.0%) and nine pregnancies in the patients assigned to IVF–embryo transfer (9/21, 42.9%), an overall clinical pregnancy rate of 32.7% (16/49; Table 4).
Table 4 Clinical pregnancy rates during the follow-up and assisted reproduction treatment periods in the therapy and control groups. Time point
Therapy group
Control group
P-value
T1 T2 T3 Cumulative pregnancy rate
6/92 (6.5) 19/86 (22.1) 16/49 (32.7) 41/92 (44.6)
0/82 (0) 4/82 (4.9) 14/58 (24.1) 18/82 (22.0)
0.030 0.001 0.390 0.002
Values are n/total (%). T1 = after 3 months of FSH therapy; T2 = 3 months after the withdrawal of therapy; T3 = after 6 months of assisted reproduction treatment.
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
8 Of the control patients who underwent assisted reproduction treatment, none had a total motile post-wash sperm count sufficient for an IUI programme, so they were enrolled only for IVF (one cycle). The clinical pregnancy rate was 24.1% (14/58), which was not significantly different from the therapy group (Table 4). In summary, 12 months after the start of the study, the cumulative clinical pregnancy rates were 44.6% (41/92) in therapy patients and 22.0% (18/82) in control patients (P = 0.002; Table 4).
Discussion Many pathological conditions can lead to oligozoospermia, such as varicocele, cryptorchidism, orchitis, testicular trauma, karyotype abnormalities, Y chromosome microdeletions, cystic fibrosis gene abnormalities, triplet amplification or polymorphism or mutation in the LH or androgen receptor genes and idiopathic causes. Indeed, reduced sperm production may be associated with different testicular alterations, such as hypospermatogenesis and maturation disturbance at different levels, most frequently involving the last spermatogenic steps (Foresta et al., 1992, 1995). In particular, spermiogenesis has a crucial role for sperm maturation. During this period, oxidative stress due to high exposure to reactive oxygen species may deeply alter mitochondrial function, DNA integrity and thus sperm competence (Baker and Aitken, 2005; O’Brien and Zini, 2005; Venkatesh et al., 2009; Zini and Libman, 2006). Moreover, some physiological conditions of different parts of the seminal tract, such as acute or chronic inflammation and infection, can affect different sperm parameters, including motility, viability and even sperm count (La Vignera et al., 2011). Thus, oligozoospermia represents the common clinical manifestation of different testicular and seminal tract alterations. It has been demonstrated that gonadotrophin therapy is highly effective in gonadotrophin-deficient men, leading to the restoration of normal spermatogenesis and spontaneous pregnancy (Bhasin, 2007; Foresta et al., 2007; Lenzi et al., 2009). However, men with normal gonadotrophin plasma concentrations can fail to respond to FSH therapy and many predictive parameters have been proposed to select responder subjects. The most frequent criteria utilized for the selection of patients is represented only by the normality of basal concentrations of gonadotrophins, while the usefulness of FSH therapy is most frequently evaluated exclusively on the basis of improvement in sperm concentration. By testicular FNAC, to clarify the particular alteration responsible for reduced sperm production, this study group previously demonstrated that FSH therapy can induce a significant increase in sperm count only when oligozoospermia was sustained by hypospermatogenesis without maturation disturbance (Foresta et al., 2002). In the presence of hypospermatogenesis associated with post-meiotic alterations, FSH further amplified the maturation difficulties in the final phases of spermatogenesis, failing to increase the sperm count (Foresta et al., 2009). Many studies have proposed evaluation of immature germ cells and particularly of spermatids, in the semen of azoospermic and severe oligozoospermic men as informative in the diagnosis of infertility (Levek-Motola
A Garolla et al. et al., 2005; Timm et al., 2005). Both cytofluorimetric and microscopic evaluation have been used for this aim (Gandini et al., 1999; Hacker-Klom et al., 1999; Yeung et al., 2007). The present study used the microscopic method suggested by WHO manual (WHO, 2010) because it is easy, rapid to perform and accessible to any seminology laboratory to test the capability of this method to identify subjects with potential responsiveness to FSH therapy. This work studied tubular function by FNAC in 174 oligozoospermic patients with normal FSH plasma concentrations and identified three subgroups: (group A) hypospermatogenesis, (group B) hypospermatogenesis with maturative disturbance and (group C) normal spermatogenesis indicating partial obstruction of seminal tract. As expected, subjects in groups A and B had significantly higher percentages of sperm aneuploidies than group C. Moreover, spermatid concentration was strongly related to tubular function, allowing this work to create a rule using spermatid concentration to diagnose hypospermatogenesis, maturative disturbance and partial obstruction with an acceptable level of accuracy. Round and elongated spermatid concentration 0.3 · 106/ml was strongly suggestive of maturative disturbance, while spermatid concentrations 0.01–0.3 · 106/ml and <0.01 · 106/ml were predictive of hypospermatogenesis and partial obstruction, respectively. As far as is known, this is the first prospective, controlled clinical study showing that evaluation of spermatid number in semen is able to predict with some accuracy tubular function in a large group of severe oligozoospermic men. In addition, to evaluate the usefulness of FSH therapy in terms of sperm parameters and clinical pregnancy rate, patients were divided into two groups: FSH therapy (patients in group A) or no therapy (patients in groups B and C). At the end of therapy, in therapy patients, the increase in FSH plasma concentration was accompanied by a significant increase in testicular volume, sperm number and motility, while in the controls, no significant variation was observed. Three months after suspension of FSH therapy, the changes in seminal parameters in therapy patients were still present. These data confirm a previous study from this study group (Foresta et al., 2000) showing that FSH therapy is effective in subjects affected by isolated hypospermatogenesis without maturation defect and suggest that spermatid count in semen is able to predict tubular status and thus response to FSH therapy. In such cases, the spermatogenic function could be efficaciously stimulated, thereby obtaining improved seminal parameters in the ejaculate. Because improvement in sperm characteristics is not often associated with improvement in clinical pregnancy rate, the primary endpoint for infertile couples, this work evaluated clinical pregnancy rate as a main outcome measurement of the effectiveness of FSH therapy. Therefore, spontaneous and assisted-conception clinical pregnancies were reported for the therapy and control groups during the whole study period: 3 months after FSH therapy (T1), 3 months after the withdrawal of therapy (T2) and 6 months after assisted reproduction treatment (T3). Previous studies evaluating the effects of FSH therapy have not found any significant increase in sperm count or pregnancy rate (Kamischke et al., 1998; Matorras et al., 1997). However, none of these studies used inclusion criteria for infertile patients strict enough to predict the response to therapy. The present
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome study detected a slight but significant increase in spontaneous pregnancies of the therapy group compared with the control group at T1 (6.5% versus 0%, P = 0.03). After the 3-month period after the withdrawal of FSH therapy, there were 19 spontaneous pregnancies in therapy subjects and four pregnancies in control subjects (22.1% and 4.9%, respectively; P = 0.001). Thereafter, all patients who had not achieved a spontaneous clinical pregnancy were recommended IUI or IVF, although it was not possible to proceed with IUI in any of the control patients due to very poor seminal parameters. In the therapy group, 49 patients underwent assisted reproduction treatment (28 by IUI and 21 by IVF) and in the control group, 58 underwent IVF treatment. Although the clinical pregnancy rate during 6 months of assisted reproduction treatment in the therapy group seemed to be higher than in the control group (32.7% versus 24.1%), the difference was not statistically significant. However, when considering the cumulative pregnancy rate for the whole study, a higher rate was detected in the therapy group compared with the control group (44.6% versus 22.0%, P = 0.002). Despite no significant reduction of aneuploidy rates and DNA fragmentation was observed, the improvement in seminal parameters induced by FSH therapy allowed some subjects to undergo assisted reproduction treatment that was less invasive for the female partners (IUI instead of IVF). Interestingly, these results were independent of the cause that induced the tubular damage. Pharmacogenetic studies have demonstrated that polymorphisms in the FSHR and FSHB genes seem to be associated with male reproductive parameters (Ferlin et al., 2011; Tu ¨ttelmann et al., 2012). On this basis, researchers have postulated that analysis of these genes could have an important role for identification of potential responders to FSH treatment (Ferlin et al., 2011; Selice et al., 2001). In conclusion, as far as is known, this is the first prospective controlled clinical study showing that evaluation of spermatid concentration in semen samples of severe oligozoospermia patients can be useful to define tubular status and to predict response to FSH therapy in terms of improved sperm parameters and increased clinical pregnancy rate. In the light of these findings, more effort should be directed at evaluating the impact of spermatid count, with the aim of improving the choice of treatment and the prospect of success in infertile patients. If these results are confirmed in further studies, patients with severe oligozoospermia and normal FSH plasma concentrations could be evaluated by noninvasive spermatid count on semen instead of testicular cytology. These findings could be particularly useful to reproduction specialists in those countries where FNAC is not routinely used. In addition, this study further demonstrates that, in selected patients, therapy with highly purified urofollitropin induces a significant improvement in sperm parameters and clinical pregnancy rate with respect to control subjects and in some cases allows couples to achieve pregnancies spontaneously or with a less invasive assisted reproduction treatment.
Acknowledgements The authors thank Alberto Bottacin, Barbara Sartini and Laura Marino for technical assistance and all the staff of
9 the Unit for Human Reproduction Pathology for helpful discussion. Special thanks to Karyn Ashley Cecchini for her kind and careful editing of English.
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11 World Health Organization, 2010. WHO Laboratory Manual for the Examination and Processing of Human Semen, fifth ed. WHO Press, Geneva, Switzerland. Yeung, C.H., Beiglbo ¨ck-Karau, L., Tu ¨ttelmann, F., Nieschlag, E., 2007. The presence of germ cells in the semen of azoospermic, cryptozoospermic and severe oligozoospermic patients: stringent flow cytometric analysis and correlations with hormonal status. Clin. Endocrinol. 67, 767–775. Zini, A., Libman, J., 2006. Sperm DNA damage: clinical significance in the era of assisted reproduction. Can. Med. Assoc. J. 175, 495–500. Declaration: The authors report no financial or commercial conflicts of interest. Received 26 September 2013; refereed 14 February 2014; accepted 18 February 2014.
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ARTICLE
Spermatid count as a predictor of response to FSH therapy Andrea Garolla a, Riccardo Selice a, Bruno Engl b, Alessandro Bertoldo a, Massimo Menegazzo a, Livio Finos c, Andrea Lenzi d, Carlo Foresta a,* a Department of Molecular Medicine, Section of Clinical Pathology and Unit for Human Reproduction Pathology, University of Padova, Italy; b Obstetrics and Gynaecology Unit, Brunico, Italy; c Department of Statistical Sciences, University of Padova, Italy; d Department of Experimental Medicine, Section of Medical Physiopathology and Endocrinology, University of Rome ‘La Sapienza’, Italy
* Corresponding author. E-mail address: [email protected] (C Foresta). Carlo Foresta is full Professor of Endocrinology and Director of Human Reproduction Pathology Unit at the University of Padova, Italy. During the last 30 years his research activities included different aspects related to male reproduction and spermatogenesis. Current research and clinical interests are in the genetics of male infertility, cryptorchidism and testicular tumours, endocrine regulation of spermatogenesis, sexual transmittable diseases and sexual disorders. He is author of more than 250 papers in International peer-reviewed journals, and invited speaker to many international and national scientific congresses. He is Past President of the Italian Society of Andrology and Sexual Medicine and member of the executive board of many scientific societies.
Abstract This study evaluated the predictive power of spermatid count and cytology for assisted reproduction outcome after FSH
therapy. A total of 174 men with severe oligozoospermia and normal plasma FSH concentration underwent semen analysis including spermatid count, TUNEL test, FISH analysis for sperm aneuploidies and testicular fine-needle aspiration cytology. Ninety-two men with hypospermatogenesis received FSH therapy for 3 months and 82 patients with maturative disturbance or partial obstruction served as controls. Semen was analysed at baseline, after FSH therapy and after 3- and 9-month follow up, and pregnancies were recorded. Subjects not reaching pregnancy at 3-month follow up were recommended assisted reproduction treatment. Spermatid count was related to testicular cytology: spermatid concentrations <0.01, 0.01–0.3 and >0.3 · 106/ml were predictive of partial obstruction, hypospermatogenesis and maturative disturbance. FSH therapy patients showed increases in sperm number and motility (both P < 0.001), allowing some couples to undergo intrauterine insemination instead of IVF. Cumulative pregnancy rate after 12 months was higher with FSH therapy (44.6%) than without (22.0%; P = 0.002). FSH therapy improved pregnancy rate and sometimes allowed less invasive assisted reproduction treatment in well-selected patients. Spermatid count could represent a new parameter to predict response to FSH therapy. RBMOnline ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Assisted reproduction outcome, FSH therapy, male infertility, pregnancy rate, severe oligozoospermia, spermatid count
http://dx.doi.org/10.1016/j.rbmo.2014.02.014 1472-6483/ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
2
Introduction Normal functioning of the gonadotrophic axis, in particular normal FSH secretion, plays crucial roles in spermatogenesis (Matsumoto, 1989; Sharpe, 1989), and several experimental and clinical studies have demonstrated the importance of FSH in regulating a normal quantitative spermatogenic process in animals and humans (Marshall et al., 1995; McLachlan et al., 1995; Moudgal et al., 1997). It has previously been demonstrated that FSH administration is able to increase sperm concentration, spermatogonal population and pregnancy rate in oligozoospermic subjects with normal plasma concentrations of gonadotrophins (Acosta et al., 1992; Bartoov et al., 1994; Glander and Kratzsch, 1997; Merino et al., 1996; Strehler et al., 1997). In addition, some authors reported that a significant improvement of sperm quality was evident after therapy. FSH therapy is useful in reducing the apoptotic process of spermatozoa and improving the qualitative properties of the axoneme, chromatin and acrosome (Baccetti et al., 1997). Recently Colacurci et al. (2012) showed that the degree of sperm DNA fragmentation significantly decreases after 90 days of FSH therapy. However, reducing fragmentation cannot guarantee fertilization and embryo development success systematically. Some authors reported that genetic, in particular epigenetic causes, can lead to abnormal spermatogenesis, with potential implications for sperm quality, fertilization and embryo development (Jenkins and Carrell, 2012; Lalancette et al., 2009). In particular, Montjean et al. (2013) evaluated methylation profiles in mature sperm DNA from normozoospermic and oligozoospermic men by observing the presence of methylation changes in spermatozoa of the latter group. Another potential mechanism of DNA damage that could be able to reduce the capability of spermatozoa to fertilize is related to DNA denaturation (Evenson and Wixon, 2008). It has been postulated that defective chromatin condensation during spermatogenesis could lead to spermatozoa unable to mature completely during spermiogenesis and therefore unable to repair DNA breaks during chromatin rearrangement occurring when histones are replaced by protamines (O’Brien and Zini, 2005; Zini and Libman, 2006). A positive effect on sperm DNA condensation after FSH therapy has been demonstrated in idiopathic infertile men (Kamischke et al., 1998). These findings may explain the increase in oocyte fertilization and pregnancy rate, sometimes in absence of improved classic seminal parameters, that has been observed in couples undergoing assisted reproduction treatment in which the male partner received FSH (Acosta et al., 1991, 1992; Ashkenazi et al., 1999). However, the improvement in seminal characteristics is variable and not much evident in many cases, and a proportion of patients do not respond to FSH therapy. Oligozoospermia may be sustained by various alterations of the seminiferous epithelium that could explain the failure of FSH therapy reported by other studies (Attia et al., 2007; Bartoov et al., 1994; Foresta et al., 1995). Fine-needle aspiration cytology (FNAC) is a minimally invasive, rapid and effective procedure to evaluate the tubular status in oligozoospermic patients (Adhikari, 2009). This study group
A Garolla et al. demonstrated that FSH therapy of oligozoospermic subjects with normal FSH plasma concentration, moderate hypospermatogenesis and absence of maturation disturbance is able to induce both a significant increase in sperm count (Foresta et al., 2002) and an improvement in pregnancy rate (Foresta et al., 2005). In contrast, when hypospermatogenesis is associated with maturative disturbance, FSH therapy shows no effect (Foresta et al., 2009). In the light of this consideration, selection criteria should be used to derive predictive information on the response to FSH therapy. Some polymorphisms in the FSH receptor gene (FSHR) are able to influence the sensitivity of the receptor for the hormone and the reproductive parameters both in men and women (Aittomaki et al., 1995; Behre et al., 2005; Greb et al., 2005; Tu ¨ttelmann et al., 2012). On this basis, the current study group tested the hypothesis that FSHR sensitivity might be important in the response to FSH therapy and demonstrated that this treatment induces diverse improvement in seminal parameters according to defined FSHR genotypes; only subjects with at least one serine in position 680 (homozygotic AS/AS and heterozygotic TN/AS) had an improvement of seminal parameters (Selice et al., 2011). Again, molecular studies on the FSHB gene (coding for the b-subunit of FSH) showed that a G/T single-nucleotide polymorphism located in the FSHB gene promoter ( 211 bp from the mRNA transcription start site; rs10835638) is responsible for the relative activity of endogenous FSH (Grigorova et al., 2007, 2008). Finally, research showed that oligozoospermic TT homozygotes for the FSHB 211 variant have a better response to FSH therapy in terms of sperm count and motility than patients with variants GG and GT (Ferlin et al., 2011). Although the pharmacogenomic approach to male infertility seems to represent an important tool to predict the response to FSH therapy, it is still very expensive, time consuming in clinical situations and requires more and larger studies to determine tailored FSH dosage. This prospective controlled clinical study aimed to identify a new, simple, cheap and effective parameter to predict the response to FSH therapy in infertile oligozoospermic patients with normal FSH plasma concentrations in terms of sperm parameters and fertility outcome.
Materials and methods Patients and setting This study was approved by the ethics committee of the Hospital-University of Padova (protocol number 2591P, approved 21 March 2010). Informed consent was obtained from each subject. All phases of the study were performed at the Infertility Centre of the University of Padova from January 2011 to December 2012. All men were informed about the FSH therapy protocol and in particular about the different phases of the study and the possibility of undergoing assisted reproduction treatment during this period. The inclusion criteria were age 25–45 years, a history of infertility for at least 2 years, sperm count <10 · 106/ml on at least three separate occasions according to World Health Organization guidelines (WHO, 2010) and any kind of infertility cause with exclusion of seminal tract infections and antisperm antibodies, normal plasma concentrations of
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome FSH (1–8 IU/l), LH (2–8 IU/l), testosterone (10–25 nmol/l) and exclusion of female factors such as ovulatory disorders, tubal factor and endocrine abnormalities evaluated by hormone assessment, pelvic ultrasound examination and hysterosalpingography. A total of 262 patients fulfilled the inclusion criteria and were enrolled in the study. Of these, 57 subjects dropped out before completing the study and 31 couples were subsequently excluded from the study because of concurrent illnesses. Therefore, 174 men affected by oligozoospermia completed the study. All patients underwent ultrasound scanning of the testis to evaluate testicular size and morphology before undergoing testicular aspiration.
Cytological and sperm analyses Fine-needle aspiration cytology All patients underwent analysis of testicular structure by means of bilateral fine-needle aspiration cytology (FNAC) to evaluate the tubular status related to the oligozoospermia. The methods of aspiration and cytological analysis have been described previously in detail (Foresta and Varotto, 1992; Foresta et al., 1992, 1995). Briefly, testicular aspiration is a simple procedure performed with a 23-gauge (0.6 mm) butterfly needle attached to a 20-ml syringe. The retrieved material was placed on two or more microscope slides for each testis, stained with May–Gru ¨nwald and Giemsa stains and examined under a light microscope at magnifications ·125, ·400 and ·1250, counting at least 200 spermatogenic cells (spermatogonia, primary and secondary spermatocytes, early and late spermatids and spermatozoa) per smear and the interposed Sertoli cells. The cell number is expressed as a percentage and represents the mean of three small aspirations performed at cranial, central and caudal level of each testis. Semen analysis Semen evaluations were performed in a blinded fashion with regard to therapy and by the same operators (WHO, 2010). Count of round and elongated spermatids was performed as suggested by the WHO manual for the quantification of immature spermatogenic cells (WHO, 2010). After liquefaction at room temperature for 30 min, 100 ll native semen was smeared on clean grease-free slides and air dried. Slides were first stained with May–Gru ¨nwald for 3 min and then rinsed twice in tap water. Thereafter, they were stained for 15 min with Giemsa (1:10 in distilled water) and then washed again three times in tap water. Spermatid count
3 was performed by light microscopy (Nikon Eclipse E600; Nikon Corporation, Japan), according to the formula shown in Figure 1. FISH analysis The study of sperm aneuploidy was performed using multicolour fluorescence in-situ hybridization (FISH), as described elsewhere (Foresta et al., 1998), for chromosomes X, Y, 13, 18 and 21. DNA hybridization was performed using two human satellite probe-specific mixes, one for chromosomes X, Y and 18 and one for chromosomes 13, 21 and 18 (Kreatech Diagnostics). Probes were directly labelled with fluorochrome PlatinumBright495 for chromosomes X and 13, resulting in a green signal, fluorochrome PlatinumBright550 for chromosomes Y and 21, resulting in a red signal, and PlatinumBright415 for chromosome 18, resulting in a blue signal. DNA denaturation of spermatozoa and probes, incubation, post-hybridization washing and nuclear staining were performed according to the manufacturer’s instructions. After preparation, slides were observed using a fluorescence microscope (Nikon Eclipse E600) equipped with a triple band-pass filter set (fluorescein isothiocyanate (FITC), tetrarhodamine isothiocyanate and 4(,6-diamidino-2-phenylindole)). Single spots were evaluated as reported elsewhere (Robbins et al., 1995). At least 2500 cells were scored for each patient. Data are expressed as the percentage of aneuploid cells. TUNEL test Evaluation of DNA fragmentation was performed as reported by Tesarik et al. (2004). The presence of sperm DNA strand breaks was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) by means of a In situ cell detection kit (Roche Diagnostics, Germany) with FITC-labelled dUTP. For each sample, 200 spermatozoa were evaluated through the fluorescent microscope with a ·100 oil-immersion objective. Results were expressed as the percentage of cells with fragmented DNA (green staining) versus normal cells (blue staining). As positive control, this study used a normal sample treated with 30 U/ml DNase A (Roche Diagnostics) in 50 mmol/l Tris-HCl pH 7.5 and 1 mg/ml BSA for 30 min at 37C to induce DNA strand breaks before labelling. The negative control was incubated in labelled solution without TdT.
Reproductive hormone analysis Plasma FSH and LH concentrations were measured in each subject by radioimmunoassay using 125I-labelled FSH and
Figure 1 (a) Formula used to determine spermatid concentration in semen samples (WHO, 2010). (b) Semen samples stained with May–Gru ¨nwald and Giemsa; arrows indicate spermatids between mature spermatozoa; bars = 10 lm. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
4 LH (Ares-Serono, Milan, Italy). The sensitivity of both the FSH and LH assays was 0.05 IU/l, as defined by mean ± 2SD multiple zero-sample measurements. Inter- and intraassay coefficients of variation were 2.8 and 3.7% for LH, and 2.6 and 3.6% for FSH, respectively. Testosterone plasma concentrations were determined in all subjects using a double-antibody radioimmunoassay commercial kit (Radim, Rome, Italy). The assay has a sensitivity of 0.1 nmol/l and cross-reacts minimally with other steroid hormones. All specimens were measured in duplicate in the same assay. Intra- and interassay coefficients of variation were 7.8 and 7.0%, respectively.
Study design After analysis at baseline (T0), subjects were studied after three consecutive phases, with primary outcomes being represented by both sperm parameters and clinical pregnancy rate: (i) 3 months after highly purified FSH therapy (T1); (ii) 3 months after the withdrawal of therapy, during which all subjects were followed for semen parameters and spontaneous pregnancies (T2); and (iii) 6 months after assisted reproduction treatment for those who had not reached clinical pregnancy during the two previous periods (T3). Figure 2 shows the flow diagram of the whole study.
A Garolla et al. Therapy After the initial assessment (history and physical examination, basal hormone parameters, semen analysis, spermatid concentration, ultrasound scanning of the testes, FNAC, FISH analysis and TUNEL test), patients were divided into three groups on the basis of their tubular function determined by FNAC: (group A) hypospermatogenesis (n = 92); (group B) hypospermatogenesis with maturative disturbance (n = 61); and (group C) normal spermatogenesis indicating partial obstruction of seminal tract (n = 21). Because hypospermatogenesis without maturative disturbance is considered a prerequisite for response to FSH therapy, patients in group A were allocated to the therapy group and patients in groups B and C were allocated to the control group. Before therapy, all patients in group A were offered preventive sperm banking at this clinic in case the therapy was ineffective or had an adverse effect. Therapy patients received 150 IU highly purified urofollitropin i.m. three times a week for 3 months and the controls did not receive any therapy. At the end of the therapy period, semen analysis, ultrasound scanning of the testes, FISH analysis and TUNEL test were repeated in all subjects by the same operators. Preand post-therapy evaluations were performed in a blinded fashion with regard to therapy. All spontaneous pregnancies
Figure 2 Flow diagram of the study. FNAC = fine-needle aspiration cytology; IUI = intrauterine insemination; IVF-ET = in-vitro fertilization embryo transfer. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome
5
occurring during this period were recorded and eventually confirmed by measurement of plasma concentrations of human chorionic gonadotrophin and by ultrasound to check the presence of a gestational sac and a fetal heart beat.
the specific indications. After this period, new pregnancies were recorded.
Follow up To evaluate the effectiveness of FSH therapy, the 174 male-factor infertile couples were followed for an additional 3-month period. All subjects were encouraged to have sexual intercourse at least two or three times per week, particularly at midcycle. All spontaneous pregnancies occurring during these 3 months were recorded. At the end of the follow-up period, all patients were re-evaluated by semen analysis.
Results are expressed as mean ± SD. Variations in FSH plasma concentrations, bi-testicular volume and motile spermatozoa between T0 and T1 are expressed as raw differences. Comparisons of groups A, B and C were performed with the ANOVA test and comparisons of the quantitative outputs of patients and controls were performed using the t-test. To evaluate the effects of FSH therapy, the twosample t-test was computed using differences with respect to T0. When the outcomes were logical (as pregnancies), Fisher’s exact test was used. The optimal discrimination rule to predict the three groups, on the basis of spermatid count, was obtained with recursive partitioning (decision tree) and the evaluation was based on cross-validated errors. The software package R (R Core Team, 2012) with package rpart were used for statistical analyses. Probability values of <0.05 were considered statistically significant.
Assisted reproduction treatment Three months after the withdrawal of FSH therapy, all couples who had not achieved spontaneous clinical pregnancy were recommended assisted reproduction treatment. For the couples who accepted treatment, those with >5 · 106/ml motile post-wash spermatozoa were enrolled to intrauterine insemination (IUI) for three cycles or IVF for one cycle (Khalil et al., 2001; Oehninger, 2000). If the number of total motile spermatozoa was not suitable for IUI (5 · 106/ml), couples were enrolled only for IVF. Either IVF–embryo transfer or intracytoplasmic sperm injection (one cycle) were performed within 6 months, considering
Statistical analysis
Results Testicular cytology obtained by FNAC and seminal smears for spermatid count from patients in each group are shown in Figure 3.
Figure 3 (a–c) Cytological images of semen obtained by fine-needle aspiration from testicles of patients with severe oligozoospermia and (d–f) seminal smears stained with May–Gru ¨nwald and Giemsa, for patients with severe hypospermatogenesis (a, d), maturative disturbance at spermatid level (b, e) and normal spermatogenesis with partial obstruction (c, f). Arrows indicate spermatids. Bars = 10 lm. Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
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A Garolla et al. Table 1 Baseline clinical, hormonal and seminal features observed in 174 oligozoospermic patients according to testicular cytology. Characteristic
Group A (n = 92)
Clinical and hormonal Age (years) Bi-testicular volume (ml) FSH (IU/l) LH (IU/l) Testosterone (nmol/l)
xxx 34.1 ± 5.9 22.8 ± 4.7a,b 5.6 ± 1.9 5.7 ± 2.3 14.1 ± 4.6 xxxx xxx 2.8 ± 1.8 5.5 ± 2.8 13.8 ± 7.6 23.7 ± 18.5c 7.4 ± 5.5 0.02 ± 0.05a,b 2.1 ± 1.0d 24.4 ± 9.5
x Seminal Semen volume (ml) Sperm concentration (· 106/ml) Sperm count (· 106) Progressive motility (%) Normal morphology (%) Spermatid concentration (· 106/ml) Aneuploidy (%) DNA fragmentation (%)
Group B (n = 61)
35.2 ± 6.0 27.3 ± 6.4 4.9 ± 1.9 5.3 ± 1.9 16.2 ± 3.5
Group C (n = 21)
32.3 ± 5.4 31.9 ± 8.6 4.4 ± 1.4 5.0 ± 2.2 15.9 ± 4.5
2.5 ± 1.1 6.1 ± 2.6 14.6 ± 7.4 27.4 ± 15.3c 8.2 ± 5.4 0.77 ± 0.67d 2.3 ± 1.2d 23.4 ± 9.3
2.1 ± 1.2 5.1 ± 2.2 12.3 ± 6.8 13.2 ± 13.6 6.2 ± 4.8 0.001 ± 0.01 1.1 ± 0.6 21.4 ± 9.9
Values are mean ± SD. Group A = hypospermatogenesis; group B = maturative disturbance; group C = partial obstruction. a Versus group B: P < 0.001. b Versus group C: P < 0.001. c Versus group C: P < 0.01. d Versus group C: P < 0.001.
Table 2 Comparison of diagnoses of hypospermatogenesis, maturative disturbance and partial obstruction by FNAC and by spermatid count, obtained by segmentation tree analysis. Diagnosis by FNAC
Group A (n = 92) Group B (n = 61) Group C (n = 21)
Diagnosis by spermatid count Hypospermatogenesis (n = 92)
Maturative disturbance (n = 61)
Partial obstruction (n = 21)
88 0 4
0 61 0
4 0 17
Values are n. Group A = hypospermatogenesis; group B = maturative disturbance; group C = partial obstruction. Hypospermatogenesis = spermatid count 0.01–0.3 · 106/ml; maturative disturbance = spermatid count 3 · 106/ml; partial obstruction = spermatid count <0.01 · 106/ml. FNAC = fine-needle aspiration cytology.
Table 1 shows the baseline clinical, hormonal and seminal characteristics observed in 174 oligozoospermic patients divided into three groups on the basis of FNAC. There were 92 subjects who showed only hypospermatogenesis (group A), 61 who showed hypospermatogenesis with maturative disturbance (44 round and 17 elongated spermatidic arrest) (group B) and 21 who showed normal spermatogenesis (group C). There were no significant differences between the three groups in terms of age, duration of infertility (3.4 ± 2.5, 3.3 ± 2.1 and 3.1 ± 2.3 years), hormonal parameters and DNA fragmentation. Patients in group A had significantly smaller bi-testicular volume (P < 0.001) than in groups B and C. Patients in groups A and B showed higher percentages of motile spermatozoa and aneuploidies than in group C, while no difference in DNA fragmentation was found. The concentration of round and elongated spematids was significantly different between the three groups
(P < 0.001). Baseline aneuploidy rate resulted significantly higher in semen form groups A and B (both P values < .0.001 versus group C).
Spermatid concentration and diagnosis of cause of oligozoospermia Using segmentation tree analysis, cross-analysing the data obtained from both the spermatid concentration and FNAC analyses allowed this study to create a rule using spermatid concentration to diagnose hypospermatogenesis, maturative disturbance and partial obstruction with an acceptable level of accuracy. Patients with spermatid concentration 0.01–0.3 · 106/ml were assigned a diagnosis of hypospermatogenesis, those with spermatid concentration 0.3 · 106/ml were assigned a diagnosis of maturative
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome disturbance and those with spermatid concentration <0.01 · 106/ml were assigned a diagnosis of partial obstruction. Table 2 shows the diagnoses of cause of oligozoospermia obtained using the spermatid concentration diagnostic rule. Among the 174 patients, correct diagnosis failed just in eight (4.6%) patients: four subjects of group A were diagnosed with partial obstruction and four patients in group C were diagnosed with hypospermatogenesis. By this method, no wrong diagnoses were reported for patients of group B.
Therapy Patients in group A (n = 92) received FSH for 3 months and patients in groups B and C (n = 82) received no FSH. FSH therapy was well tolerated in all subjects, and no significant side effects were reported. No variation in seminal volumes was observed in the therapy and control groups (Table 3). In the therapy group, there were significant improvements in sperm concentration and count after therapy, with a doubling of sperm concentration with respect to T0 and the control group (both P <0.001; Table 3). The therapy group had also a significant increase of plasma FSH (from 5.6 ± 1.9 to 10.4 ± 2.3 IU/l), bi-testicular volume (from 22.8 ± 4.7 to 23.2 ± 4.6 ml) and progressively motile spermatozoa (from 2.8 ± 2.1 to 9.6 ± 5.4 · 106/ml) between T0 and T1 (P < 0.001, P < 0.05 and P < 0.001, respectively). No significant variations were observed in other seminal parameters, such as normal morphology (from 7.4 ± 5.5 to 8.3 ± 5.2%), sperm aneuploidies (from 2.1 ± 1.0 to 2.0 ± 1.1%), DNA fragmentation (from 24.4 ± 9.5 to 24.1 ± 8.2%),
Table 3 Seminal parameters throughout the study in the therapy and control groups. Parameter
Therapy group (n = 92)
Seminal volume (ml) T0 T1 T2 T3
xxx 2.9 ± 1.8 2.9 ± 1.8 2.9 ± 1.8 2.9 ± 1.8 xxxx xxx
x Sperm concentration (· 106/ml) T0 T1 T2 T3
x Sperm count (· 106) T0 T1 T2
5.5 ± 2.8 11.7 ± 5.4a 9.9 ± 4.1a 5.9 ± 3.1 xxxx xxx 13.8 ± 7.6 28.2 ± 15.9a 24.1 ± 10.7a
Control group (n = 82)
2.6 ± 1.1 2.5 ± 1.1 2.6 ± 1.1 2.6 ± 1.0
5.5 ± 2.4 5.6 ± 3.4 5.4 ± 3.5 4.7 ± 3.8
13.9 ± 7.1 14.3 ± 9.3 13.1 ± 9.1
Values are mean ± SD. T0 = at baseline; T1 = after 3 months of FSH therapy; T2 = 3 months after the withdrawal of therapy; T3 = after 6 months of assisted reproduction treatment. a Versus T0 and controls: P < 0.001.
7 plasma LH (from 5.7 ± 2.3 to 5.2 ± 2.2 IU/l) and testosterone concentration (from 14.1 ± 4.6 to 14.3 ± 4.3 nmol/l). In the control group, no significant differences were observed after therapy (Table 3). For T0 and T1, bitesticular volume from 28.5 ± 7.3 to 28.3 ± 7.4 ml, plasma FSH from 4.9 ± 1.8 to 5.0 ± 1.7 IU/l, plasma LH from 5.2 ± 2.0 to 5.4 ± 1.9 IU/l, testosterone from 16.1 ± 3.7 to 16.0 ± 3.6 nmol/l, progressively motile spermatozoa from 2.9 ± 2.0 to 3.1 ± 1.9 · 106, normal morphology from 7.7 ± 5.3 to 8.4 ± 5.6%, sperm aneuploidies from 1.9 ± 1.2 to 2.0 ± 1.1% and DNA fragmentation from 22.9 ± 9.4 to 23.5 ± 9.1%). During the therapy period, spontaneous pregnancies were observed only in the therapy group (6/92 versus 0/82 couples, P = 0.03).
Follow up Three months after the withdrawal of FSH therapy, another sperm analysis was performed in all subjects. The therapy group showed significantly higher sperm concentration and count with respect to T0 that were not different from those observed at T1 (P < 0.001; Table 3). After the follow-up period, the therapy group showed a significantly higher clinical pregnancy rate than the control group, with 19/86 (22.1%) patients achieving spontaneous pregnancies in the treated group versus 4/82 (4.9%) in the control group (P = 0.001; Table 4).
Assisted reproduction treatment Six months after the withdrawal of FSH therapy (T3), sperm analysis did not show any significant difference in seminal parameters between therapy and control patients (Table 3). During this period, assisted reproduction treatment was recommended to the patients who had not achieved pregnancy. In the therapy group, patients accepting assisted reproduction treatment were enrolled in two treatment programmes: 28 patients to IUI (three cycles) and 21 subjects to IVF–embryo transfer (one cycle). After 6 months of treatment, seven pregnancies were achieved in patients assigned to IUI (7/28, 25.0%) and nine pregnancies in the patients assigned to IVF–embryo transfer (9/21, 42.9%), an overall clinical pregnancy rate of 32.7% (16/49; Table 4).
Table 4 Clinical pregnancy rates during the follow-up and assisted reproduction treatment periods in the therapy and control groups. Time point
Therapy group
Control group
P-value
T1 T2 T3 Cumulative pregnancy rate
6/92 (6.5) 19/86 (22.1) 16/49 (32.7) 41/92 (44.6)
0/82 (0) 4/82 (4.9) 14/58 (24.1) 18/82 (22.0)
0.030 0.001 0.390 0.002
Values are n/total (%). T1 = after 3 months of FSH therapy; T2 = 3 months after the withdrawal of therapy; T3 = after 6 months of assisted reproduction treatment.
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
8 Of the control patients who underwent assisted reproduction treatment, none had a total motile post-wash sperm count sufficient for an IUI programme, so they were enrolled only for IVF (one cycle). The clinical pregnancy rate was 24.1% (14/58), which was not significantly different from the therapy group (Table 4). In summary, 12 months after the start of the study, the cumulative clinical pregnancy rates were 44.6% (41/92) in therapy patients and 22.0% (18/82) in control patients (P = 0.002; Table 4).
Discussion Many pathological conditions can lead to oligozoospermia, such as varicocele, cryptorchidism, orchitis, testicular trauma, karyotype abnormalities, Y chromosome microdeletions, cystic fibrosis gene abnormalities, triplet amplification or polymorphism or mutation in the LH or androgen receptor genes and idiopathic causes. Indeed, reduced sperm production may be associated with different testicular alterations, such as hypospermatogenesis and maturation disturbance at different levels, most frequently involving the last spermatogenic steps (Foresta et al., 1992, 1995). In particular, spermiogenesis has a crucial role for sperm maturation. During this period, oxidative stress due to high exposure to reactive oxygen species may deeply alter mitochondrial function, DNA integrity and thus sperm competence (Baker and Aitken, 2005; O’Brien and Zini, 2005; Venkatesh et al., 2009; Zini and Libman, 2006). Moreover, some physiological conditions of different parts of the seminal tract, such as acute or chronic inflammation and infection, can affect different sperm parameters, including motility, viability and even sperm count (La Vignera et al., 2011). Thus, oligozoospermia represents the common clinical manifestation of different testicular and seminal tract alterations. It has been demonstrated that gonadotrophin therapy is highly effective in gonadotrophin-deficient men, leading to the restoration of normal spermatogenesis and spontaneous pregnancy (Bhasin, 2007; Foresta et al., 2007; Lenzi et al., 2009). However, men with normal gonadotrophin plasma concentrations can fail to respond to FSH therapy and many predictive parameters have been proposed to select responder subjects. The most frequent criteria utilized for the selection of patients is represented only by the normality of basal concentrations of gonadotrophins, while the usefulness of FSH therapy is most frequently evaluated exclusively on the basis of improvement in sperm concentration. By testicular FNAC, to clarify the particular alteration responsible for reduced sperm production, this study group previously demonstrated that FSH therapy can induce a significant increase in sperm count only when oligozoospermia was sustained by hypospermatogenesis without maturation disturbance (Foresta et al., 2002). In the presence of hypospermatogenesis associated with post-meiotic alterations, FSH further amplified the maturation difficulties in the final phases of spermatogenesis, failing to increase the sperm count (Foresta et al., 2009). Many studies have proposed evaluation of immature germ cells and particularly of spermatids, in the semen of azoospermic and severe oligozoospermic men as informative in the diagnosis of infertility (Levek-Motola
A Garolla et al. et al., 2005; Timm et al., 2005). Both cytofluorimetric and microscopic evaluation have been used for this aim (Gandini et al., 1999; Hacker-Klom et al., 1999; Yeung et al., 2007). The present study used the microscopic method suggested by WHO manual (WHO, 2010) because it is easy, rapid to perform and accessible to any seminology laboratory to test the capability of this method to identify subjects with potential responsiveness to FSH therapy. This work studied tubular function by FNAC in 174 oligozoospermic patients with normal FSH plasma concentrations and identified three subgroups: (group A) hypospermatogenesis, (group B) hypospermatogenesis with maturative disturbance and (group C) normal spermatogenesis indicating partial obstruction of seminal tract. As expected, subjects in groups A and B had significantly higher percentages of sperm aneuploidies than group C. Moreover, spermatid concentration was strongly related to tubular function, allowing this work to create a rule using spermatid concentration to diagnose hypospermatogenesis, maturative disturbance and partial obstruction with an acceptable level of accuracy. Round and elongated spermatid concentration 0.3 · 106/ml was strongly suggestive of maturative disturbance, while spermatid concentrations 0.01–0.3 · 106/ml and <0.01 · 106/ml were predictive of hypospermatogenesis and partial obstruction, respectively. As far as is known, this is the first prospective, controlled clinical study showing that evaluation of spermatid number in semen is able to predict with some accuracy tubular function in a large group of severe oligozoospermic men. In addition, to evaluate the usefulness of FSH therapy in terms of sperm parameters and clinical pregnancy rate, patients were divided into two groups: FSH therapy (patients in group A) or no therapy (patients in groups B and C). At the end of therapy, in therapy patients, the increase in FSH plasma concentration was accompanied by a significant increase in testicular volume, sperm number and motility, while in the controls, no significant variation was observed. Three months after suspension of FSH therapy, the changes in seminal parameters in therapy patients were still present. These data confirm a previous study from this study group (Foresta et al., 2000) showing that FSH therapy is effective in subjects affected by isolated hypospermatogenesis without maturation defect and suggest that spermatid count in semen is able to predict tubular status and thus response to FSH therapy. In such cases, the spermatogenic function could be efficaciously stimulated, thereby obtaining improved seminal parameters in the ejaculate. Because improvement in sperm characteristics is not often associated with improvement in clinical pregnancy rate, the primary endpoint for infertile couples, this work evaluated clinical pregnancy rate as a main outcome measurement of the effectiveness of FSH therapy. Therefore, spontaneous and assisted-conception clinical pregnancies were reported for the therapy and control groups during the whole study period: 3 months after FSH therapy (T1), 3 months after the withdrawal of therapy (T2) and 6 months after assisted reproduction treatment (T3). Previous studies evaluating the effects of FSH therapy have not found any significant increase in sperm count or pregnancy rate (Kamischke et al., 1998; Matorras et al., 1997). However, none of these studies used inclusion criteria for infertile patients strict enough to predict the response to therapy. The present
Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014
Spermatid count as a predictor of FSH therapy outcome study detected a slight but significant increase in spontaneous pregnancies of the therapy group compared with the control group at T1 (6.5% versus 0%, P = 0.03). After the 3-month period after the withdrawal of FSH therapy, there were 19 spontaneous pregnancies in therapy subjects and four pregnancies in control subjects (22.1% and 4.9%, respectively; P = 0.001). Thereafter, all patients who had not achieved a spontaneous clinical pregnancy were recommended IUI or IVF, although it was not possible to proceed with IUI in any of the control patients due to very poor seminal parameters. In the therapy group, 49 patients underwent assisted reproduction treatment (28 by IUI and 21 by IVF) and in the control group, 58 underwent IVF treatment. Although the clinical pregnancy rate during 6 months of assisted reproduction treatment in the therapy group seemed to be higher than in the control group (32.7% versus 24.1%), the difference was not statistically significant. However, when considering the cumulative pregnancy rate for the whole study, a higher rate was detected in the therapy group compared with the control group (44.6% versus 22.0%, P = 0.002). Despite no significant reduction of aneuploidy rates and DNA fragmentation was observed, the improvement in seminal parameters induced by FSH therapy allowed some subjects to undergo assisted reproduction treatment that was less invasive for the female partners (IUI instead of IVF). Interestingly, these results were independent of the cause that induced the tubular damage. Pharmacogenetic studies have demonstrated that polymorphisms in the FSHR and FSHB genes seem to be associated with male reproductive parameters (Ferlin et al., 2011; Tu ¨ttelmann et al., 2012). On this basis, researchers have postulated that analysis of these genes could have an important role for identification of potential responders to FSH treatment (Ferlin et al., 2011; Selice et al., 2001). In conclusion, as far as is known, this is the first prospective controlled clinical study showing that evaluation of spermatid concentration in semen samples of severe oligozoospermia patients can be useful to define tubular status and to predict response to FSH therapy in terms of improved sperm parameters and increased clinical pregnancy rate. In the light of these findings, more effort should be directed at evaluating the impact of spermatid count, with the aim of improving the choice of treatment and the prospect of success in infertile patients. If these results are confirmed in further studies, patients with severe oligozoospermia and normal FSH plasma concentrations could be evaluated by noninvasive spermatid count on semen instead of testicular cytology. These findings could be particularly useful to reproduction specialists in those countries where FNAC is not routinely used. In addition, this study further demonstrates that, in selected patients, therapy with highly purified urofollitropin induces a significant improvement in sperm parameters and clinical pregnancy rate with respect to control subjects and in some cases allows couples to achieve pregnancies spontaneously or with a less invasive assisted reproduction treatment.
Acknowledgements The authors thank Alberto Bottacin, Barbara Sartini and Laura Marino for technical assistance and all the staff of
9 the Unit for Human Reproduction Pathology for helpful discussion. Special thanks to Karyn Ashley Cecchini for her kind and careful editing of English.
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Please cite this article in press as: Garolla, A et al. Spermatid count as a predictor of response to FSH therapy. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.014