JOURNAL TRANSCRIPT

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RBMOnline - Vol 15. No 2. 2007 156-160 Reproductive BioMedicine Online; www.rbmonline.com/Article/2798 on web 14 June 2007

Article Gonadotrophin therapy in combination with ICSI in men with hypogonadotrophic hypogonadism M Emre Bakircioglu received his MD degree from Cerrahpasa Medical School, Istanbul University. He concluded his urology residency in Haydarpasa Numune Hospital and then completed a research fellowship in neuro-urology and erectile dysfunction under supervision of Dr Tom Lue at the Department of Urology, University of California San Francisco. He is currently working at the German Hospital Urology Department, and is also a consulting urologist at the German Hospital IVF Centre and Bahçeci Women Healthcare Center. He is member of American Urology Association, American Society for Reproductive Medicine and Society for Male Reproduction and Urology.

Dr Emre Bakircioglu Mustafa Emre Bakircioglu1,3, Halit Firat Erden2, H Nadir Çiray2, Numan Bayazit2, Mustafa Bahçeci2 1 German Hospital Urology Department; 2German Hospital IVF Centre and Bahçeci Women Healthcare Centre, Istanbul, Turkey 3 Correspondence: Cesmebasi Cad. Caliskan Sok. Kemerhill Sitesi, Madra 1/9, Kemerburgaz, Eyup, Istanbul, Turkey, 34077. Tel: +90 212 2932150; Fax: +90 212 2443050; e-mail: [email protected]

Abstract The aim of this study was to evaluate the impact of gonadotrophin therapy in combination with intracytoplasmic sperm injection (ICSI) in men with hypogonadotrophic hypogonadism (HH). Twenty-five azoospermic men were diagnosed with HH due to low FSH, LH and total testosterone concentrations. These patients were treated with human chorionic gonadotrophin for 1 month plus recombinant FSH the following month. Total testosterone concentrations were measured in the first and third months. Semen analyses were performed monthly after the third month of treatment. ICSI was performed when sperm production commenced. Total testosterone concentration and testicular volume were significantly increased after gonadotrophin therapy (P < 0.001). On average, spermatozoa were detected in the ejaculate after 10 months. Spontaneous pregnancies were achieved in four couples. Twenty-two ICSI cycles were performed in 18 couples using ejaculated or testicular spermatozoa, and 12 pregnancies (54.5% per cycle) were achieved. These results showed that HH could be treated successfully with hormonal therapy combined with ICSI using ejaculated spermatozoa. The use of ICSI made it possible to achieve pregnancy when spermatozoa appeared in the ejaculate, and shortened the duration of gonadotrophin therapy. Keywords: azoospermia, gonadotrophin therapy, hypogonadotrophic hypogonadism, intracytoplasmic sperm injection, testicular sperm extraction

Introduction

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Azoospermia due to hypogonadotrophic hypogonadism (HH) is an uncommon cause of male infertility. HH is categorized as primary or secondary. Primary HH is also known as idiopathic HH (IHH), and is a disorder that selectively affects the secretion or function of gonadotrophin-releasing hormone (GnRH). As a result, LH and FSH are not produced by the hypophysis and therefore neither androgen production nor spermatogenesis is stimulated in the testes. Secondary HH may indicate the presence of various underlying diseases such as brain tumour, infiltrative disorders (sarcoidosis, haemochromatosis, infection) or head trauma.

The stimulation of spermatogenesis can be successfully achieved either with pulsatile administration of GnRH or a combination of human chorionic gonadotrophin (HCG)/human menopausal gonadotrophin (HMG) in the infertility treatment of HH (Buchter et al., 1998). Continuous HCG alone may result in the presence of spermatozoa in the ejaculate (Vicari et al., 1992). Instead of using urinary menotropin preparations, the recombinant human FSH (r-hFSH) has been successfully used for the induction of spermatogenesis and fertility in gonadotrophin-deficient men (Liu et al., 1999).

© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

Article - A treatment for hypogonadotrophic hypogonadism - ME Bakircioglu et al.

Spontaneous pregnancies have been reported after prolonged durations of hormonal therapy in IHH patients (Kliesch et al., 1994; Buchter et al., 1998; Liu et al., 1999). Sperm appearance in the semen can be expected 6 months after the initiation of gonodotrophin therapy, and pregnancy can be predicted on average 8 months after sperm concentration increases to 5 × 106/ml (Buchter et al., 1998; Liu et al., 1999). Therefore, it was reported that if spontaneous pregnancy does not occur after 20 months, or 8 months after achieving a sperm concentration of 5 × 106/ml, assisted reproductive technologies may be considered time effective (Liu et al., 2002). The combination of hormonal therapy with intracytoplasmic sperm injection (ICSI) has been reported in a few studies (Liu et al., 1999; Fahmy et al., 2004; Zorn et al., 2005). Despite undergoing prolonged gonodotrophin therapy, some patients with HH remained azoospermic. It was reported that in 11 out of 15 (73%) men with HH who were still azoospermic after gonadotrophin therapy, spermatozoa were successfully retrieved from testicular tissue (Fahmy et al., 2004). Using testicular spermatozoa in 17 ICSI cycles, the pregnancy rate was 20%. The aim of this study was to evaluate the effect of the combination of HCG and r-hFSH therapy plus ICSI of ejaculated spermatozoa on the occurrence of pregnancy in the partners of men with IHH.

Materials and methods Patients Between 2002 and 2005, 25 patients (who were not referred from endocrinology or other clinics) were diagnosed with HH due to azoospermia and low concentrations of FSH, LH and total testosterone. The ejaculate volumes of the patients were lower than 1 ml due to the low testosterone concentrations. The mean age of the patients and their partners were 34.5 ± 5.2 (mean ± SD) years and 31.2 ± 4.2 years respectively. The duration of infertility was 7.1 ± 4.0 years. Azoospermia was confirmed after two to three semen analyses and examination of the pellet suspension subsequent to centrifugation at 600 g. All of the patients had a history of decreased libido and sexual dysfunction. Additionally in 15 patients, facial, axillary and pubic hair was decreased and gynaecomastia was observed. Three patients had anosmia. The testicular volume was measured by a Prader orchidometer (Accurate Surgical and Scientific Instruments Corporation, NY, USA) at the beginning and after 6 months of therapy. Testicular volumes were ≤4 ml in 19 and >4 ml in six patients (range 5–8 ml). Medical history and pituitary imaging by magnetic resonance imaging did not show any evidence of acquired (secondary) hypogonadotrophic hypogonadism in patients with testicular volume >4 ml. One patient had right cryptorchidism at the level of the external inguinal ring. Ten patients had a history of testosterone replacement therapy (TRT) and eight patients had a history of gonadotrophin therapy (HCG alone or combination with urinary FSH-LH). These patients discontinued the therapy for 4 weeks and then testosterone concentration was measured before gonadotrophin therapy in this study was initiated. In two patients (11% of the patients who accepted ICSI therapy), testicular sperm extraction (TESE) was performed, as there were no motile spermatozoa in their thawed material and in their ejaculate on the day of oocyte retrieval. RBMOnline®

Gonadotrophin therapy Patients started gonadotrophin therapy with 5000 IU i.m. HCG (Pregnyl; Organon, Oss The Netherlands) twice weekly. The dose of HCG was adjusted to once a week according to the testosterone concentration during follow-up visits. One month after the initiation of HCG, recombinant human FSH (GonalF; Industria Farmaceutica Serono S.p.A., Bari, Italy) 100 IU was administered as subcutaneous injection three times a week. The serum testosterone concentrations were determined in the first and the third month of therapy and semen analyses were performed monthly after the third month of gonadotrophin therapy.

Sperm preservation When motile spermatozoa were presented in the ejaculate, two or three semen samples were collected in order to cryopreserve spermatozoa before the ICSI cycle started. A semen sample was collected again on the day of oocyte retrieval and cryopreserved spermatozoa were thawed if the motile spermatozoa did not suffice to inject all metaphase II oocytes.

Testicular sperm extraction (TESE) Under general anaesthesia, the scrotum was incised on the scrotal raphe. The testis was opened from the mid part with a large horizontal incision under ×10 magnification using an operating microscope. Microdissection procedure and testicular sperm preparation were performed as previously described (Bakircioglu et al., 2006).

Ovarian stimulation A pelvic genital examination and transvaginal ultrasonography were performed on partners of HH patients who were scheduled to undergo ICSI treatment. Routine laboratory tests, including FSH and prolactin, were carried out. The standard long protocol with agonist desensitization with leuprolide acetate (Lucrin daily; Abbott, Turkey) was started on day 21 of the previous menstrual cycle. For poor responders, flare-up protocol was performed as previously described (Akman et al., 2001). When at least two follicles reached 18 mm in diameter, 10,000 IU HCG (Pregnyl; Organon, Oss, The Netherlands) was administered intramuscularly. Oocytes were retrieved under general anaesthesia 34–36 h later and were subjected to ICSI. Fertilization was assessed 16–18 h after ICSI. Embryos were cultured at 37°C, in an atmosphere of 5.5% CO2 in air in individual 30 μl drops of a human tubal fluid based medium (Sage In-Vitro Fertilization Inc., CT, USA) covered with mineral oil. The embryos were selected according to day-3 embryo quality. The luteal phase was supported by 100 mg/day progesterone in oil i.m. Clinical pregnancy was defined as a demonstrable gestational sac by transvaginal ultrasonography, subsequent to a rise in β-HCG concentrations.

Statistical analysis Mann−Whitney U-test was used to compare the initial testicular volume and testosterone concentrations with those after 6 months of therapy and the time of sperm appearance in the ejaculate of

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Article - A treatment for hypogonadotrophic hypogonadism - ME Bakircioglu et al. men with ≤4 ml and >4 ml testicular volume. Fisher’s exact test was performed to analyse the pregnancy rates in the partners of men with ≤4 ml and >4 ml testicular volume. A P-value 0.05).

Four spontaneous pregnancies occurred during gonadotrophin therapy. One pregnancy was achieved after 18 months of therapy at a sperm concentration of 11 × 106/ml, 6 months after unsuccessful ICSI treatment. Three pregnancies were achieved after 12 months of therapy at sperm concentrations of 6 × 106/ ml, 5 × 106/ml and 1 × 106/ml. The volume of the testes was smaller than 4 ml in three patients and 8 ml in one patient who achieved pregnancy after an unsuccessful ICSI treatment. The pregnancy rates achieved for patients who had testicular volume larger than 4 ml and smaller than 4 ml were 66.7 and 63.2% respectively and the difference was not statistically significant. Twenty-two ICSI cycles were performed on 18 patients and in one patient transfer of frozen−thawed embryos was performed. Three patients did not undergo ICSI treatment. The distribution of sperm concentrations and the duration of the gonadotrophin therapy in those patients who underwent ICSI therapy are shown in Table 2. A total of 331 oocytes were collected, of which 261 were in metaphase II. Of these, 169 (65%) were fertilized. A mean of 3.0 ± 0.8 embryos were transferred. Twelve clinical pregnancies

Table 1. Clinical characteristics of patients with hypogonadotrophic hypogonadism during gonadotrophin therapy and intracytoplasmic sperm injection (ICSI) outcome.

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Patient

Age Months of (years) treatment before sperm presence

Sperm count Total testosterone (ng/ml) Testicular volume (ml) Pregnancy (× 106/ml) Basal 1 month 3 months Pre6 months treatment

1 2 3 4 5 6 7d 8 9e 10 11 12 13 14 15 16 17e 18e 19d 20 21 22 23e 24 25

40 36 32 36 35 39 33 32 39 30 27 22 41 28 43 31 37 39 30 32 33 41 42 31 33

0.1 0.1 0.1 PP PP PP PP PP PP PP PP 10.0 0.5 0.3 0.1 PP PP PP PP PP PP PP PP PP PP

7 12 12 10 11 11 6 6 8 7 11 12 12 6 12 8 10 11 12 10 8 18 9 11 9

0.3 0.3 0.2 0.9 0.2 0.2 1.1 0.5 0.5 0.7 0.2 1.0 0.9 0.3 1.3 0.3 0.3 0.5 0.3 0.3 0.2 0.1 0.2 0.4 0.3

2.70 2.80 2.23 3.50 2.82 3.39 3.27 3.42 1.99 2.70 1.49 2.00 3.66 3.72 1.73 2.50 3.70 4.10 3.00 3.27 3.79 2.80 2.40 3.57 2.99

4.50 4.80 2.38 3.20 4.65 3.30 4.58 5.50 7.70 4.85 3.37 5.65 7.52 6.82 6.80 4.40 4.29 5.78 4.40 4.60 5.64 5.50 3.50 4.51 7.03

5 5 6 7 8 8 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 4 4 4 4

6 8 8 10 12 10 4 4 4 4 4 4 4 4 4 5 5 5 5 4 4 5 6 5 6

Yes Yes No Yes Yesa No Yes Yes No No Yes Yesa Yes Yesa Yes Yesa Yes No Yesb No ICSI No ICSI No Yesc No ICSI Yes

PP = pellet positive. a Spontanous pregnancy, bfrozen−thawed embryo transfer, cmissed abortions, dsperm recovery with testicular sperm extraction, etwo ICSI attempts. RBMOnline®

Article - A treatment for hypogonadotrophic hypogonadism - ME Bakircioglu et al. Table 2. Intracytoplasmic sperm injection (ICSI) results in patients with hypogonadotrophic hypogonadism according to sperm concentration. Parameter

Sperm concentration Pellet +