2.1 Patients

We retrospectively evaluated the microscopic TESE and ICSI outcomes of hypogonadotropic hypogonadal patients who remained azoospermic after gonadotropin therapy between 2004 and 2024. Patients were eligible if they had hormonal findings consistent with hypogonadotropic hypogonadism, available pituitary magnetic resonance imaging (MRI) to rule out hypothalamic or pituitary abnormalities, and persistent azoospermia despite receiving gonadotropin therapy for at least six months. The study received approval from our institutional review board.

2.2 Clinical and laboratory evaluation

All patients underwent a comprehensive assessment, including a detailed medical history and a thorough physical examination. Azoospermia was confirmed by at least two semen analyses. Seminal parameters were assessed according to the criteria established by the World Health Organization (WHO), with azoospermia defined as the complete absence of spermatozoa following centrifugation of the semen at 3000g for 15 minutes and a thorough examination of the pellet [6].

HH was defined as low morning total testosterone in conjunction with low or inappropriately normal FSH and luteinizing hormone (LH) levels. In borderline cases, free testosterone was calculated using sex hormone-binding globulin and albumin levels. Pituitary MRI was performed in all cases to identify any structural lesions that might present and contribute to HH [7].

2.3 Treatment protocol

Therapeutic intervention was initiated with either urinary hCG (Pregnyl 5000 IU) or recombinant hCG (Ovitrelle 250 μg), administered subcutaneously twice weekly, corresponding to a total weekly dose of approximately 3000–5000 IU. Treatment was maintained for 24 weeks, during which patients were monitored every three months with semen analysis and hormonal profiling, and the dose was titrated as needed to achieve optimal serum testosterone levels. Semen samples were obtained via masturbation following 3–7 days of sexual abstinence. In cases where spermatogenesis was not restored with hCG monotherapy, highly purified human menopausal gonadotropin (hMG) (Menopur 75 IU) was subsequently added to the regimen three times per week. During combined gonadotropin therapy, the frequency of hCG administration and the dosage of hMG were gradually adjusted based on periodic hormone measurements. Treatment was continued until an adequate therapeutic response was achieved, with patients counseled that the appearance of spermatozoa in the ejaculate typically required 6 to 12 months of gonadotropin therapy.

2.4 Microscopic TESE procedure

Microscopic TESE was performed under sedoanalgesia and local anaesthesia according to the technique previously described by Schlegel [8]. Informed consent was obtained from all patients prior to the procedures. The obtained seminiferous tubule samples were transferred to a sterile Petri dish containing 3 mL of HEPES buffered medium and dissected according to the mechanical technique [9]. Microscopic examination of the wet preparation performed by experienced embryologists was carried out at ×400 magnification under an inverted microscope (Olympus IX71, Olympus Corporation, Tokyo, Japan). In cases where sperm could not be retrieved from the dissected testis, the same procedure was applied to the contralateral testis.

In cases where sperm was retrieved, the final sample was combined with the same volume of sperm freezing medium (CryoSperm, Origio, Måløv, Denmark). The resulting mixture was then immersed in liquid nitrogen for storage at −196 °C.

In all patients, a sample of seminiferous tubules was immersed in Bouin’s solution and subsequently sent for histopathological evaluation.

2.5 Ovarian stimulation and ICSI

Controlled ovarian hyperstimulation was conducted using either the long protocol or the antagonist protocol, as previously described [10]. Oocyte retrieval was performed 36 hours after hCG administration. Following denudation, metaphase II (MII) oocytes were prepared for ICSI procedure. Routine ICSI was performed after a 2 to 2.5-hour incubation period, as previously described by Palermo et al. [11]. After 16 to 18 hours, embryos were assessed for fertilization and subsequently for cleavage. Daily evaluations of embryo cleavage were conducted until the transfer day. Embryos were graded according to the grading system of ALPHA Scientists Special Interest Group [12]. Embryo transfers were performed 3 to 5 days post-ICSI, following four to six days of luteal support in the frozen embryo transfer cycle.

2.6 Data interpretation

The patients were categorized into two groups based on the presence of spermatozoa during micro-TESE. Group I comprised patients from whom spermatozoa were successfully retrieved, while Group II consisted of patients from whom no spermatozoa could be obtained. Age, HH etiology, ejaculate volume, serum hormone levels at the start of the treatment, type and duration of gonadotropin treatment prior to micro-TESE, the laterality of the micro-TESE procedure (unilateral and bilateral) and the results of histopathological evaluations were compared between groups.

To evaluate the outcomes of ICSI, we assessed the fertilization rate (FR), implantation rate (IR), clinical pregnancy fertilized embryo rate (CPR), and live birth rate (LBR) for those who underwent the procedure. FR is defined as the ICSI. IR was calculated as the percentage of s (mean number of 2 pronuclear embryos) relative to the total number of MII oocytes that underwent he number of gestational sacs observed in the uterine cavity per transferred embryo. CPR was defined as the presence of a fetal heartbeat at least two weeks following the hCG test. Live birth was defined as the delivery of a baby after 24 weeks of gestation. The cumulative CPR refers to the pregnancy rate across both fresh and frozen-thawed embryo transfer (ET) cycles.

2.7 Statistical analysis

Kolmogorov-Smirnov test or Shapiro-Wilk test was used to assess the normality of continuous variable distributions. Normally distributed continuous variables were presented as mean ± standard deviation, while non-normally distributed continuous variables were reported as median. Depending on their distribution, continuous variables were compared using either Student’s t-test for parametric data or the Mann-Whitney U test for non-parametric data. The categorical variables between the groups were analyzed by using the Chi square test or Fisher’s Exact test. Statistical significance was defined as a p-value of less than 0.05. Statistical analysis was conducted using Statistical Package for the Social Science (SPSS) version 26.0 (SPSS Inc., Chicago, IL, USA).

3.1 Patient characteristics

There were 17 patients who became azoospermic after gonadotropin treatment due to HH. Only one of these patients had HH secondary to pituitary adenoma surgery, while the others had isolated HH. Baseline characteristics of patients with a mean age of 34.4 ± 5.2 years are summarized in Table 1.

While 10 patients received urinary HCG-based treatment, 7 patients received recombinant HCG-based treatment. There was no statistically significant difference between the treatment types and treatment durations of the patients (Table 1).

Unilateral micro-TESE was applied to 5 patients and bilateral microscopic TESE was applied to 12 patients, there was no statistical difference between the groups (p = 0.515).

3.2 Micro-TESE results

While sperm was obtained from 14 patients (82.4%) after gonadotropin treatment, there was no statistically significant difference in terms of the interpreted data between the group in which sperm was obtained and the group in which it was not obtained (Table 1).

Histopathological examination revealed varying degrees of maturation arrest in all patients (Table 1).

3.3 ICSI, embryological and clinical outcomes

ICSI was performed using frozen-thawed sperm in 12 of these 14 patients from whom sperm was obtained surgically. The remaining 2 patients did not start ICSI treatment. The mean age of the partners was 27 ± 7.1 years. A total of 25 good quality embryos were transferred in 18 transfer cycles in these patients. The implantation rate was calculated as 28%. Six of these cycles resulted in clinical pregnancy, and four of them resulted in delivery. Three of the pregnancies were singletons and one was twins. Embryological and clinical results are summarized in Table 2 (Ref. [5, 13, 14, 15, 16, 17]).