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  • Clinical Study Reduced Seminal Concentration of CD45pos Cells after Follicle-Stimulating Hormone Treatment in Selected Patients with Idiopathic Oligoasthenoteratozoospermia

    Rosita A. Condorelli,1 Aldo E. Calogero,1 Enzo Vicari,1 Laura Mongioi’,1 Giovanni Burgio,1

    Rossella Cannarella,1 Filippo Giacone,1 Linda Iacoviello,1

    Giuseppe Morgia,2 Vincenzo Favilla,2 Sebastiano Cimino,2 and Sandro La Vignera1

    1 Department of Medical and Pediatric Sciences, Section of Endocrinology, Andrology and Internal Medicine, University of Catania, Policlinico “G. Rodolico”, Building 4, Rm 2C18, Via S. Sofia 78, 95123 Catania, Italy

    2 Department of Urology, University of Catania, Catania, Italy

    Correspondence should be addressed to Sandro La Vignera;

    Received 11 September 2013; Revised 20 November 2013; Accepted 20 November 2013; Published 14 January 2014

    Academic Editor: Muhammad Shahab

    Copyright © 2014 Rosita A. Condorelli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    The present study evaluated the conventional sperm parameters and the seminal concentration of CD45pos cells (pan-leukocyte marker) of infertile patients with idiopathic oligoasthenoteratozoospermia (OAT). The patients were arbitrarily divided into three groups treated with recombinant follicle-stimulating hormone FSH: 𝛼 (Group A = 20 patients), recombinant FSH-𝛽 (Group B = 20 patients), and highly purified human FSH (Group C = 14 patients). All treated groups achieved a similar improvement of the main sperm parameters (density, progressive motility, and morphology), but only the increase in the percentage of spermatozoa with normal morphology was significant compared to the baseline in all three examined groups. Moreover, all groups had a significant reduction of the seminal concentration of CD45pos cells and of the percentage of immature germ cells. Before and after the treatment, the concentration of CD45pos cells showed a positive linear correlation with the percentage of immature germ cells and a negative correlation with the percentage of spermatozoa with regular morphology. These results demonstrate that treatment with FSH is effective in patients with idiopathic OAT and that there are no significant differences between the different preparations. The novelty of this study is in the significant reduction of the concentration of CD45pos cells observed after the treatment.

    1. Introduction

    Follicle-stimulating hormone has an important role in testic- ular function. In particular, its main biological actions (medi- ated through a receptor systempresent on the Sertoli cells) are the following: cellular sperm differentiation, modulation of spermatid morphogenesis, and the maturation of epididymal spermatozoa [1–10].

    The biological actions of FSH are more relevant in the clinical condition of hypogonadotropic hypogonadism [5, 11–16] and are more controversial in functional hypogo- nadism and/or in patients with idiopathic oligoasthenoter- atozoospermia (OAT) [17–27]. Functional hypogonadism is defined as a clinical condition in which the gonadotropin

    levels are in the low-normal range but the testosterone level is inadequate (or often in the low-normal range) [28]. Idiopathic OATmeans that no etiological factor can be found by common clinical, instrumental, or laboratory methods [29].

    The demonstrated biological effects of FSH treatment in male infertile patients are increased spermatogonial popu- lation and sperm count, [17–21], increased rate of fertiliza- tion, and increased pregnancy rate in programs of assisted fertilization [22–24]. However, several studies show a lack of effects of FSH on sperm parameters [25–27].

    The Italian Medicines Agency authorizes the use of preparations of FSH for the treatment of male infertility in males with hypogonadism and in infertile males with low or

    Hindawi Publishing Corporation International Journal of Endocrinology Volume 2014, Article ID 372060, 8 pages

  • 2 International Journal of Endocrinology

    normal levels of FSH, but not more than 8mIU/mL in any case.

    There are, however, lesser known aspects relating to the possible usefulness of FSH therapy with FSH on uncon- ventional as well as conventional sperm parameters (den- sity, morphology, and motility). For example, FSH therapy could induce modifications that could restore the quality of the inflammatory response of the semen. Under partic- ular conditions, the leukocyte concentration in semen is directly correlated with immature germ cell concentrations and the percentage of abnormal forms [30]. The defects of spermatogenesis represent a potential target of hormonal treatment as well as a potential cause of increased inflam- matory response, in particular the response of leukocytes, the main mediators of this phenomenon. Together with their classic antimicrobial action, leukocytes also remove immature germ cells and/or sperm with maturational defects [31–33], suggesting they have a protective role in semen at concentrations of

  • International Journal of Endocrinology 3

    the presence of low resistance, a prolonged systolic phase, flow maintenance during diastole, and a low resistance index (IR: 0.62). Systolic flow speed along the centripetal arteries was considered normal if it was lower than 15 cm/sec and/or between 4 and 12 cm/sec [41–43].

    2.3. Hormone Measurements. Hormonal evaluations were performedby electrochemiluminescencewithHitachi-Roche equipment (Cobas 6000, Roche Diagnostics, Indianapolis, IN, USA). The reference intervals were as follows: LH = 1.6– 9.0mIUmL−1, FSH = 2.0–12.0mIUmL−1, 17𝛽-estradiol = 8.0–43.0 pgmL−1, total testosterone = 2.8–8.0 ngmL−1, and prolactin = 4.0–15.0 ngmL−1. Blood sampling was performed at 8:00 am, after at least 8 hours of sleep. The determination of serum LH and prolactin was repeated after an interval of 30 minutes.

    2.4. Sperm Analysis. Semen samples were collected by mas- turbation into a sterile container after 2–7 days of sexual abstinence and were transported to the laboratory within 30 minutes after ejaculation. According to the 2010 WHO guidelines, each sample was evaluated for seminal volume, pH, sperm count, progressive motility, morphology, and round cell concentration [37]. For all patients, the sperm analysis was repeated after 4 months, at the end of the hormonal treatment.

    2.5. Conventional Measurement of Seminal Leukocytes and Immature Germ Cells

    2.5.1. Seminal Leukocytes (Peroxidase Test). The protocol used was adapted from that of Endtz [44]. The working solu- tion used for the test was obtained by adding 1 𝜇L of H

    2 O 2 to

    20𝜇L of a 0.09% 3,3󸀠-diaminobenzidine tetrahydrochloride stock solution (DAB, ISOPAC, Sigma, Milan, Italy) in 40% ethanol. In each assay, 20𝜇L of semen was incubated with 20𝜇L of working solution in an Eppendorf tube for 5minutes at room temperature. Before setting up the slide, 40 𝜇L of PBS was added. Peroxidase-positive cells were marked by yellow-brown-red staining, while peroxidase-negative cells remained colorless. At least 100 round cells were counted using an optical microscope at 400x magnification, and the percentages of peroxidase-positive and -negative cells were evaluated. The total leukocyte count is expressed in millions per milliliter of semen.

    2.5.2. Immature Germ Cell Evaluation in Semen. Spermatids were differentiated from leukocytes by a seminal fluid smear stained using the Papanicolaou technique [45]. Spermatids were identified on the basis of the following parame- ters: coloration, size, core shape and size (approximately 5 𝜇M), absence of intracellular peroxidase, and the absence of leukocyte-specific antigen (see Section 2.6). Morpholog- ically, multinucleated spermatids were distinguished from polymorphonuclear leukocytes by the presence of a pink color in contrast to the bluish color of polymorphonuclear leukocytes.

    2.6. Cytofluorimetric Assessment. Theanalysis was conducted with an EPICS XL Flow Cytometer (Coulter Electronics, IL, USA), equipped with an argon laser at 488 nm and three fluorescence detectors: green (FL-1 at 525 nm), orange (FL- 2 to 575 nm), and red (FL-3 at 620 nm). For each sample, 100,000 events were measured at a low flow velocity and analyzed using System II, version 3.0.

    2.6.1. Flow-Cytometric Analysis of CD45pos Cells. To perform the absolute lymphocyte count, 100 𝜇L of each liquefied semen sample was incubated with a mixture containing Syto-16 green fluorescent nucleic acid stain to identify the spermatozoa and exclude debris (final concentration 200 nM) (Molecular Probes, Eugene, Oregon, USA), 7- amino-actinomycin D (7-AAD, Via-Probe, BD Pharmingen, San Diego, CA, USA) to assess viability, anti-CD45-APC (pan-leukocyte antigen) to recognize white blood cells, and anti-CD16-PE to PMN recognize. The addition of 100 𝜇L of Flow-Count Fluorospheres (Beckmann-Coulter, Fullerton, CA, USA) at 1034 beads/mL allowed us to determine the absolute lymphocyte count by flow cytometry. After incuba- tion in the dark for 20 minutes at room temperature, 1mL of PBS was added, and the sample was analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA). For each test, 100,000 events were acquired. The total number of leukocytes permilliliterwas calculated by applying the following formula [39]:

    % monoclonal antibody − (positive cells × no. of spermatozoa/mL) 100



    2.7. Different Hormonal Treatments Analyzed. Sperm pa