Article Gonadotrophin therapy in combination with ICSI in men with hypogonadotrophic hypogonadism

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RBMOnline - Vol 15. No 2. 2007 156-160 Reproductive BioMedicine Online; www.rbmonline.

1 RBMOnline - Vol 15. No Reproductive BioMedicine Online; 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 Bakircioglu 1,3, Halit Firat Erden 2, H Nadir Çiray 2, Numan Bayazit 2, Mustafa Bahçeci 2 1 German Hospital Urology Department; 2 German 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, Tel: ; Fax: ; ebakircioglu@yahoo.com 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 156 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) Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 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 /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 /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. 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 (Gonal- F; 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 h later and were subjected to ICSI. Fertilization was assessed h after ICSI. Embryos were cultured at 37 C, in an atmosphere of 5.5% CO 2 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 157

3 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 was considered statistically significant. Statistics Package for Social Sciences version 11.5 for windows software (serial no: ) was used for statistical analysis. Results At the beginning of therapy, the mean testicular volume of the patients was 3.7 ± 1.9 ml and the mean total testosterone concentration was 0.5 ± 0.3 ng/ml. Testosterone concentration was significantly increased to 3.0 ± 0.7 ng/ml in the first month and to 5.1 ± 1.2 ng/ml in the third month (P < 0.001). Testicular volume was significantly increased and reached 5.7 ± 2.3 ml by the sixth month (P < 0.001). Spermatozoa were detected in the ejaculate in all patients at a mean of 10.1 ± 2.6 months of therapy (range 6 18 months) (Table 1). Spermatozoa appeared in the ejaculate at 10.5 ± 1.8 months in men with testicular volume larger than 4 ml and 9.7 ± 2.9 months in men with testicular volume smaller than 4 ml (P > 0.05). Four spontaneous pregnancies occurred during gonadotrophin therapy. One pregnancy was achieved after 18 months of therapy at a sperm concentration of /ml, 6 months after unsuccessful ICSI treatment. Three pregnancies were achieved after 12 months of therapy at sperm concentrations of / ml, /ml and /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. Patient Age Months of Sperm count Total testosterone (ng/ml) Testicular volume (ml) Pregnancy (years) treatment ( 10 6 /ml) Basal 1 month 3 months Pre- 6 months before sperm treatment presence Yes Yes No PP Yes PP Yes a PP No 7 d 33 6 PP Yes PP Yes 9 e 39 8 PP No PP No PP Yes Yes a Yes Yes a Yes PP Yes a 17 e PP Yes 18 e PP No 19 d PP Yes b PP No ICSI PP No ICSI PP No 23 e 42 9 PP Yes c PP No ICSI PP Yes 158 PP = pellet positive. a Spontanous pregnancy, b frozen thawed embryo transfer, c missed abortions, d sperm recovery with testicular sperm extraction, e two ICSI attempts.

4 Table 2. Intracytoplasmic sperm injection (ICSI) results in patients with hypogonadotrophic hypogonadism according to sperm concentration. Parameter Sperm concentration Pellet + <10 5 /ml /ml /ml No. of ICSI cycles No. of pregnancies 2 a Mean duration of the 10.3 ± ± ± ± 2.8 therapy (months) a Both resulted in abortion. Two cycles in which testicular sperm extraction was applied are not included. (54.5% per cycle, 67% per patient) were achieved and two consecutive ICSI cycles were performed in four couples. In one couple, the two pregnancies resulted in missed abortions. In two couples, no pregnancy was achieved by either ICSI attempt. In the remaining couple, pregnancy was achieved at the second attempt. In addition, in one couple frozen thawed embryo transfer resulted in a pregnancy. Six pregnancies resulted in deliveries of eight boys and three girls, and five pregnancies are ongoing. Discussion The aetiology of idiopathic HH is not fully understood; however, recent studies suggest that the pathology may have a genetic and molecular basis (Wolczynski et al., 2003; Paduch et al., 2005; Pengo et al., 2006). In the treatment of men with HH, GnRH or HCG, with or without FSH, can successfully initiate spermatogenesis (Finkel et al., 1985; Schopohl et al., 1991; Buchter et al. 1998). Although different protocols have been used in the treatment of HH, prolonged continuous therapy, followup with the assessment of hormone concentrations and semen analyses are mandatory to achieve successful sperm production. Usually, the protocols start with HCG and after stabilization of the serum testosterone concentration, FSH is then included in the protocol (European Metrodin HP Study Group 1998; Liu et al., 1999; Bouloux et al., 2002). The efficacy of using recombinant or urinary FSH was reported to be similar (Liu et al., 2002). Spermiogenesis and sperm presence in the ejaculate occurred in response to HCG and r-hfsh therapy at a mean of 10 months in the present study group. This result compares with a mean of 9 months in HCG combined with highly purified urinary FSH treatment (European Metrodin HP Study Group, 1998). However, in some studies 13 53% of men with HH failed to produce any spermatozoa after treatment (Burris et al., 1988; Schopohl et al., 1991; Burgues and Calderon, 1997; European Metrodin HP Study Group, 1998; Liu et al., 2002). TESE could be an option for men with HH who still have azoospermia after a long period of therapy. In one study, it is reported that in 11 of 15 patients (73%) spermatozoa were successfully recovered after TESE (Fahmy et al., 2004). In this study, in two of 18 (11%) patients, who had occasional spermatozoa in the pellet, no spermatozoa were detected in the ejaculate on the day of oocyte retrieval. Spermatozoa were successfully recovered from testicular tissue in those patients. Several semen samples were collected and cryopreserved in order to avoid the TESE operation; however, motile spermatozoa could not be recovered on the day of oocyte retrieval. Therefore, in consultations for ICSI treatment, men who have occasional spermatozoa in the pellet should be informed of the possibility of the TESE procedure and their treatment could be postponed for several months to increase sperm concentration in the ejaculate. Gonadotrophin therapy is mandatory to induce spermatogenesis and testicular development in men with HH who seek fertility. It was shown that testicular volume was the most prominent clinical factor to predict pregnancy and spermatogenesis in those patients (Liu et al., 2002). Patients who had a testicular volume larger than 4 ml were generally agreed to have a better prognosis in terms of pregnancy (Burris et al., 1988; Vicari et al., 1992; Liu et al., 2002). In this study, the time of sperm appearance and pregnancy rates were not statistically significantly different between those patients who had larger and smaller than 4 ml testicular volume. In addition, three of the patients who had <4 ml testicular volume achieved spontaneous pregnancy. All of the patients who achieved spontaneous pregnancy had at least /ml sperm concentration after 12 months of therapy. It is reported that pregnancies mostly occurred within 8 months after achieving /ml sperm concentrations (Liu et al., 2002). In the progress of infertility treatment of patients who have had at least 1 year of therapy and sperm concentrations of < /ml, or who have > /ml sperm concentration and in whose partner a pregnancy does not occur after 20 months, assisted reproductive technologies may be suggested to the couple. In addition to the factors above, the ovarian reserve and the age of the partner should be considered. ICSI is an alternative infertility treatment in men with HH, and the results of ICSI with gonadotrophin therapy have been presented in several studies in the literature. Yong et al. (1997) reported an ICSI attempt in one patient with post-pubertal onset of HH after 9 months of gonadotrophin therapy. Another study showed an ICSI pregnancy using cryopreserved spermatozoa and frozen thawed embryo transfer that resulted in a miscarriage (Liu et al., 1999). Zorn et al. (2005) reported four men with HH who underwent ICSI after 6 23 months of gonadotrophin therapy. Ten ICSI cycles were performed and resulted in a 67% fertilization rate and 30% pregnancy rate per cycle (Zorn et al., 2005). In the present study, fertilization of the oocytes was 65% and the pregnancy rate was 54.5% in 22 ICSI attempts. So far as 159

5 is known, this is the highest number of patients with HH using ejaculated spermatozoa for ICSI. These results suggest that ICSI may be considered as the choice of infertility treatment in men with HH, even though the sperm concentrations are very low in their ejaculate. Further studies need to be conducted to assess the effect of sperm concentrations on ICSI results in the treatment of men with HH. In conclusion, gonadotrophin therapy for men with HH should be for at least a year, and during the therapy patients need support and careful assessment. The results show that pregnancy rates are promising with ICSI. Hence, during the gonadotrophin therapy, ICSI can be considered as a treatment option, and therefore might be discussed with patients who have commenced sperm production. References Akman MA, Erden HF, Tosun SB et al Comparison of agonistic flare-up-protocol and antagonistic multiple dose protocol in ovarian stimulation of poor responders: results of a prospective randomized trial. Human Reproduction 16, Bakircioglu ME, Erden HF, Kaplancan T et al Aging may adversely affect testicular sperm recovery in patients with Klinefelter syndrome. Urology 68, Bouloux P, Warne DW, Loumaye E 2002 Efficacy and safety of recombinant human follicle-stimulating hormone in men with isolated hypogonadotropic hypogonadism. Fertility and Sterility 77, Buchter D, Behre HM, Kliesch S et al Pulsatile GnRH or human chorionic gonadotropin/human menopausal gonadotropin as effective treatment for men with hypogonadotropic hypogonadism: a review of 42 cases. European Journal of Endocrinology 139, Burgues S, Calderon MD 1997 Subcutaneous self-administration of highly purified follicle stimulating hormone and human chorionic gonadotrophin for the treatment of male hypogonadotrophic hypogonadism. Spanish Collaborative Group on Male Hypogonadotropic Hypogonadism. Human Reproduction 12, Burris AS, Rodbard HW, Winters SJ et al Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size. Journal of Clinical Endocrinology and Metabolism 66, European Metrodin HP Study Group 1998 Efficacy and safety of highly purified urinary follicle-stimulating hormone with human chorionic gonadotropin for treating men with isolated hypogonadotropic hypogonadism. Fertility and Sterility 70, Fahmy I, Kamal A, Shamloul R et al ICSI using testicular sperm in male hypogonadotrophic hypogonadism unresponsive to gonadotrophin therapy. Human Reproduction 19, Finkel DM, Phillips JL, Snyder PJ 1985 Stimulation of spermatogenesis by gonadotropins in men with hypogonadotropic hypogonadism. New England Journal of Medicine 313, Kliesch S, Behre HM, Nieschlag E 1994 High efficacy of gonadotropin or pulsatile gonadotropin-releasing hormone treatment in hypogonadotropic hypogonadal men. European Journal of Endocrinology 131, Liu PY, Gebski VJ, Turner L et al Predicting pregnancy and spermatogenesis by survival analysis during gonadotrophin treatment of gonadotrophin-deficient infertile men. Human Reproduction 17, Liu PY, Turner L, Rushford D et al Efficacy and safety of recombinant human follicle stimulating hormone (Gonal-F) with urinary human chorionic gonadotrophin for induction of spermatogenesis and fertility in gonadotrophin-deficient men. Human Reproduction 14, Paduch D A, Mielnik A, Schlegel P N 2005 Novel mutations in testisspecific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reproductive BioMedicine Online 10, Pengo M, Ferlin A, Arredi B et al FSH receptor gene polymorphisms in fertile and infertile Italian men. Reproductive BioMedicine Online 13, Schopohl J, Mehltretter G, von Zumbusch R et al Comparison of gonadotropin-releasing hormone and gonadotropin therapy in male patients with idiopathic hypothalamic hypogonadism. Fertility and Sterility 56, Vicari E, Mongioi A, Calogero AE et al Therapy with human chorionic gonadotrophin alone induces spermatogenesis in men with isolated hypogonadotrophic hypogonadism long-term follow-up. International Journal of Andrology 15, Wolczynski S, Laudanski P, Jarzabek K et al A case of complete hypogonadotropic hypogonadism with a mutation in the gonadotropin-releasing hormone receptor gene. Fertility and Sterility 79, Yong EL, Lee KO, Ng SC et al Induction of spermatogenesis in isolated hypogonadotrophic hypogonadism with gonadotrophins and early intervention with intracytoplasmic sperm injection. Human Reproduction 12, Zorn B, Pfeifer M, Virant-Klun I et al Intracytoplasmic sperm injection as a complement to gonadotrophin treatment in infertile men with hypogonadotrophic hypogonadism. International Journal of Andrology 28, Received 20 February 2007; 8 March 2007; accepted 25 May

EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 Gonadotropin or GnRH treatment in hypogonadal men 299 The patients with hypothalamic disorders (group I)

European Journal of Endocrinology (1998) 139 298 303 ISSN 0804-4643 Pulsatile GnRH or human chorionic gonadotropin/human menopausal gonadotropin as effective treatment for men with hypogonadotropic hypogonadism: