Predictive factors shaping fertility potential and reproductive outcomes in men with Klinefelter syndrome

Abstract

Klinefelter syndrome (KS), marked by testicular heterogeneity, provides a rationale for testicular sperm extraction in azoospermic men, yet large-scale evaluations of predictive and reproductive outcomes remain limited. This retrospective study included 79 karyotype-confirmed KS patients who underwent conventional testicular sperm extraction (cTESE) and microdissection testicular sperm extraction (micro-TESE) between February 2001 and August 2024. Clinical, hormonal, histological, embryological, and reproductive data were analyzed, with successful sperm retrieval (SR) defined as the presence of at least one spermatozoon. SR was achieved in 40.5% of patients and was significantly associated with larger testicular volumes (left: P = 0.018; right: P = 0.028), lower follicle-stimulating hormone (mean ± standard deviation [s.d.]: 29.06 ± 14.49 IU l⁻¹ vs 38.22 ± 16.97 IU l⁻¹; P = 0.016), and lower luteinizing hormone levels (mean ± s.d.: 16.41 ± 7.46 IU l⁻¹ vs 20.65 ± 9.59 IU l⁻¹; P = 0.047). These parameters also correlated with the number of frozen sperm vials. Among those with SR, 46 embryo transfer cycles were conducted, involving 79 embryos. Fertilization, cleavage, and blastulation rates were 50.3%, 80.7%, and 40.2%, respectively. Good-quality embryos were observed in 45.1% on day 3 and 55.0% on day 5, with clinical pregnancy and live birth rates of 39.1% and 39.5% per transfer. These findings indicate that favorable hormonal profiles and testicular characteristics are predictive of SR success and that reproductive outcomes among KS patients with sperm retrieval are comparable to those in non-KS populations. Overall, the results support the continued use of testicular sperm extraction as a viable path to genetic parenthood in this population.

INTRODUCTION

Klinefelter syndrome (KS), a genetic condition that affects males, typically results from an extra X chromosome (47,XXY karyotype).1 It is the most common chromosomal aneuploidy leading to decreased fertility potential, affecting one in 660 men.2 KS is associated with various physical, developmental, and reproductive issues, including reduced muscle mass, metabolic syndrome, bone mass loss, enlarged breast tissue, tall stature, and infertility characterized by hypergonadotropic hypogonadism.2,3 Individuals with KS might also experience cognitive impairment including learning difficulties and speech delays.4

While testicular function deteriorates progressively,5 approximately two-thirds of patients will maintain normal testosterone levels until the second and third decades of life.6 As a result, many cases remain undiagnosed until adulthood, often becoming apparent only when couples seek infertility counseling.7,8

This delayed diagnosis is clinically significant, as patients with non-obstructive azoospermia (NOA) may still harbor residual foci of spermatogenesis amid atrophic, hyalinized testicular tissue.9 Therefore, mature spermatozoa can be detected in testicular biopsies.10 This testicular heterogeneity is the rationale for offering KS patients microsurgical sperm extraction, typically followed by intracytoplasmic sperm injection (ICSI).5 According to the recent data, the sperm retrieval rate (SRR) in these procedures ranges between 43% and 50%.5,11,12

Significant effort is dedicated to identifying more precise predictive factors affecting chances for sperm retrieval in KS patients such as anti-Müllerian hormone (AMH) levels and hormonal assays.12,13,14 Although most studies suggest that fertility preservation is ideally pursued between the end of the second and third decades of life,15,16,17,18 Renault et al.12 recently concluded that delaying these procedures does not impair the SRR.

To date, large-scale studies evaluating predictive factors for positive sperm retrieval (SR) in conventional testicular sperm extraction (cTESE) and microdissection testicular sperm extraction (micro-TESE) procedures of KS patients remain limited.12,14,16,17 Studies assessing fertilization outcomes among those with successful SR are even fewer and typically involve smaller cohorts.11,13,19,20,21,22,23

Our study aims to address these gaps by refining predictive factors for successful SR in KS patients undergoing cTESE and micro-TESE procedures for KS patients and by presenting comprehensive embryological and reproductive outcomes among those with successful retrieval. These findings will better inform KS patients about their chances of SR and their potential for biological parenthood.

PATIENTS AND METHODS

Study population

We conducted a retrospective cohort study at a single university-affiliated tertiary medical center (Institute for the Study of Fertility & Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center affiliated to the Faculty of Medicine and Health Sciences, Tel Aviv University, Tel Aviv, Israel). The inclusion criteria were as follows: (1) men with a confirmed diagnosis of KS established via karyotype analysis following referral for infertility evaluation and (2) men had undergone either cTESE or micro-TESE between January 2001 and June 2024. All patients were referred to Institute for the Study of Fertility & Racine IVF Unit for the evaluation of infertility. The sample size reflected all eligible KS patients treated during this period; no formal power calculation was performed. Patients’ data were collected from the electronic medical records (EMRs). For positive-SR group, fertilization and embryological data were collected from the female partner’s EMRs, and missing information was supplemented via telephone questionnaires; therefore, informed consent was obtained. The study was approved by the Institutional Review Board of Tel Aviv Sourasky Medical Center affiliated to the Faculty of Medicine and Health Sciences (IRB TLV-0369-24), including approval for public data sharing.

Demographic, clinical embryologic and reproductive data

The two primary outcomes were positive SR and the number of straws that were frozen following cTESE/micro-TESE. A positive-SR was defined as identification of at least 1 spermatozoon following the procedure. Collected variables included demographic, clinical, and reproductive characteristics including patient age, history of cryptorchidism or varicocele, procedure type (cTESE or micro-TESE), testicular volume measured by orchidometer, hormonal profiles (follicle-stimulating hormone [FSH], luteinizing hormone [LH], testosterone, and prolactin) obtained within 3 months prior to the procedure, and presurgical Y-chromosome microdeletion data.

Fertilization rate was defined as the proportion of injected oocytes that developed 2 pronuclei (2PN) following ICSI. Cleavage rate was defined as the proportion of 2PN zygotes that developed into cleavage stage embryos. Blastulation rate was defined as the proportion of 2PN zygotes reaching the blastocyst stage. The implantation rate was calculated by the number of gestational sacs observed divided by the number of embryos transferred.24

Embryo morphology was assessed according to the Society for Assisted Reproductive Technology (SART) criteria for cleavage-stage embryos25 and the Gardner grading system for blastocysts.26 Clinical pregnancy rate was defined as the number of clinical pregnancies per embryo transfer cycle. Live birth rate was defined as the number of live births per embryo transfer cycle.24

Sperm extraction

Both cTESE and micro-TESE procedures were performed under general anesthesia. A longitudinal incision was made along the scrotal raphe, followed by careful separation of connective tissue to expose the testicle. A horizontal incision in the tunica albuginea was then performed to access the testicular parenchyma.

In cTESE, a small biopsy sample (approximately 50–150 mg) was excised and immediately placed in modified human tubal fluid (mHTF) medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 5% human serum albumin (HSA).

In micro-TESE, the procedure began with a wide midline opening of the tunica albuginea to optimize visualization of the seminiferous tubules while preserving testicular blood supply. Under high magnification (20×–25×) using an operating microscope (Zeiss OPMI® Vario S88; Prescott’s Surgical Microscopes, Monument, CO, USA), larger and more opaque seminiferous tubules were selectively identified, as they were more likely to contain spermatozoa.27 Small biopsy samples (approximately 2–5 mg) were excised from these targeted tubules, along with an additional standard biopsy taken from adjacent areas for histological evaluation.

As surgical practice advanced, cTESE was gradually replaced by mTESE, with the majority of mTESE procedures taking place from 2014 onward.

Tissue processing

Upon arrival at the laboratory, tissue specimens were finely minced using two sterile 25-gauge needles attached to syringes and transferred into a test tube containing modified HTF medium supplemented with 5% HSA. The resulting suspension was gently vortexed for approximately 10 s to facilitate cell dispersion. Immediately after vortexing, the supernatant was carefully aspirated, avoiding the residual tissue fragments. The collected supernatant was then centrifuged to concentrate cellular elements. A 5-µl aliquot of the centrifuged suspension was examined using a wet mount under phase-contrast microscopy to identify spermatozoa. Additionally, twelve individual 5-µl drops were evaluated under inverted microscopy to enhance detection sensitivity.

Sperm cryopreservation

If viable spermatozoa were detected, aliquots of the sperm suspension were prepared for cryopreservation. To determine the number of vials to freeze, a representative sample was microscopically examined to estimate the total number of viable spermatozoa, assuming approximately 50% attrition during the freeze–thaw process.The suspension was then aliquoted such that each vial contained approximately 40–50 viable spermatozoa, intended for future ICSI.

Histology assessment

Histological analysis was conducted on Bouin’s-fixed, paraffin-embedded testicular biopsy specimens stained with hematoxylin and eosin (H&E). Histopathological classification was based on the most advanced spermatogenic cell type observed through combined histological and cytological evaluation. Biopsy specimens were categorized into two distinct patterns: (1) mixed atrophy (MA), characterized by the coexistence of tubules displaying Sertoli cell-only (SCO) patterns alongside tubules containing spermatogenesis up to mature spermatozoa and (2) SCO pattern, defined by the exclusive presence of Sertoli cells without any detectable germ cells within the seminiferous tubules. A minimum of 20 seminiferous tubules were evaluated per specimen. The initial classification was performed in the andrology laboratory of the Institute for the Study of Fertility & Racine IVF Unit and subsequently verified by the pathology department of the Tel Aviv Sourasky Medical Center affiliated to the Faculty of Medicine and Health Sciences.

Fertilization and embryo handling

Ovarian stimulation was induced by one of the three standard protocols: (1) a short gonadotropin-releasing hormone (GnRH) agonist; (2) fixed GnRH antagonist; or (3) mid-luteal long GnRH agonist. Ovulation was triggered by 250 mcg of human recombinant chorionic gonadotrophin (Choriogonadotropin Alfa; Ovitrelle, Merck Serono, Italy) or 0.2 mg of a GnRH agonist (Triptorelin; Ferring Pharmaceuticals, Saint-Prex, Switzerland) when at least two follicles reached a mean diameter of at least 17 mm.

Oocyte retrieval was performed 36–38 h after triggering ovulation, using ultrasound-guided transvaginal aspiration. ICSI was performed in all the cycles using a Nikon inverted microscope (Diaphot 300; Nikon, Tokyo, Japan) with Narishige micromanipulators (Narishige, Tokyo, Japan). Embryos were individually cultured in an EmbryoSlide culture dish embedded with human embryo culture medium (SAGE 1-Step™single-step medium (CooperSurgical, Trumbull, CT, USA) covered with paraffin oil (Oil for Embryo Culture; Irvine Scientific) to allow individual assessment and documentation. Incubation in an EmbryoScope incubator (5% oxygen, 5.5% carbon dioxide and temperature of 37°C) started immediately after ICSI and continued up to day 5 or 6 of development. The embryo cleavage rate and morphology were evaluated on day 3 and continued until day 5 or 6 prior to embryo transfer or cryopreservation.

Statistical analyses

Patients’ characteristics and fertilization outcomes in the positive-SR group were summarized with descriptive statistics. Normally distributed quantitative variables were described using mean ± standard deviation (s.d.) and non-normally distributed variables using median (interquartile range [IQR]). Histograms, Q-Q plots, and Kolmogorov–Smirnov tests determined distribution normality. Univariate analysis techniques identified group differences. Continuous variables’ significance was assessed using Student’s t-test or Mann–Whitney U test. Similarly, we used Chi-square or Fisher’s exact for the categorical variables. Linear correlations were assessed using Pearson correlation coefficient. We employed recursive partitioning analysis using a classification and regression tree (CART) approach in order to create decision trees that split the dataset into subgroups based on variables that best differentiated between positive and negative SR outcomes. The resulting tree highlighted key variables and cut-off points associated with positive SR, providing an interpretable clinical decision framework. P < 0.05 was considered statistically significant. Multivariable associations were further assessed using nominal logistic regression to estimate the odds ratios (ORs) and adjust for potential confounders. R version 4.4.1 (R project, Vienna, Austria) was used to analyze the data. All analyses were conducted on a fully de-identified database, with no personally identifiable information available to the researchers.

RESULTS

During the study period, 79 procedures (44 cTESE and 35 micro-TESE), were performed for patients at our center encompassing a wide range of ethnicities within the Israeli population (Supplementary Table 1). SR was unsuccessful in 47 patients (59.5%) and successful in 32 patients (40.5%), resulting in a SRR of 40.5%. The patient age (mean ± s.d.) was 32.2 ± 6.2 years, and the female partners’ age (mean ± s.d.) was 28.9 ± 4.4 years. Four procedures were unilateral, and the remaining were bilateral. Patients with positive SR had larger testicular volumes and lower LH and FSH levels compared to those with unsuccessful retrieval. Y-chromosome microdeletion testing was negative in all the patients included in this cohort.

Supplementary Table 1.

Ethnic origin of positive and negative sperm-retrieval Klinefelter syndrome patients

Origin	Positive sperm-retrieval	Negative sperm-retrieval
Sephardic Jews, n (%)	8 (53.3)	5 (35.7)
Ashkenazi Jews, n (%)	3 (20.0)	4 (28.6)
Mixed ethnicity*, n (%)	3 (20.0)	2 (14.3)
Arab, n (%)	1 (6.7)	3 (21.4)
Total, n (%)	15 (100)	14 (100)

^*Mixed ethnicity refers to individuals of both Sephardic and Ashkenazi origin. Ethnic background was partially collected

Table 1 summarizes the demographic, clinical, laboratory, and histological findings of the cohort. No statistically significant differences were observed in baseline characteristics such as age, history of cryptorchidism or varicocele, KS mosaicism, or the type of TESE procedure performed (all P > 0.05). However, patients in the successful SR group exhibited larger left and right testicular volumes (P = 0.018 and P = 0.028, respectively), lower FSH and LH levels (P = 0.016 and P = 0.047, respectively), and a higher prevalence of MA histology compared to SCO histology in the retrieved specimens (P < 0.001).

Table 1.

Comparison of baseline characteristics, laboratory, and histology findings of Klinefelter (KS) syndrome patients

Parameter	Positive sperm retrieval	Negative sperm retrieval	P
Total numbers, n (%)	32 (40.5)	47 (59.5)	-
Age (year), mean±s.d.	31.9±7.0	32.4±5.7	0.751
Partner’s age (year), mean±s.d.	29.0±5.1	28.9±3.7	0.559
TESE, n (%)			0.935
cTESE	18 (56.3)	26 (55.3)
Micro-TESE	14 (43.8)	21 (44.7)
Side of operation, n (%)			0.058
Left	1 (3.1)	0 (0)
Right	3 (9.4)	0 (0)
Bilateral	28 (87.5)	47 (100.0)
Left testicular size (cm), median (IQR)	2.0 (1.5–2.5)	1.5 (1.5–2.0)	0.018*
Right testicular size (cm), median (IQR)	2.0 (1.5–2.5)	1.5 (1.5–2.0)	0.028*
S/P cryptorchidism, n (%)	4 (12.5)	6 (12.8)	0.972
S/P varicocele, n (%)	1 (3.1)	3 (6.4)	0.517
Mosaicism, n (%)	3 (9.4)	3 (6.4)	0.622
Histology, n (%)			<0.001*
SCO	11 (34.4)	41 (87.2)
MA	21 (65.6)	6 (12.8)
FSH (IU l−1), mean±s.d.	29.06±14.49	38.22±16.97	0.016*
LH (IU l−1), mean±s.d.	16.41±7.46	20.65±9.59	0.047*
Testosterone (nmol l−1), mean±s.d.	11.90±5.25	10.56±6.15	0.328
Prolactin (mIU l−1), mean±s.d.	297.21±227.15	236.51±129.99	0.235

^*Statistical significance at the P<0.05 level. TESE: testicular sperm extraction; cTESE: conventional testicular sperm extraction; micro-TESE: microdissection testicular sperm extraction; SCO: Sertoli cells only; MA: mixed atrophy; FSH: follicle-stimulating hormone; s.d.: standard deviation; IQR: interquartile range, LH: luteinizing hormone; -: not applicable

Predictors of successful SR

Using multivariable forward stepwise logistic regression, two independent factors were associated with successful SR: lower LH levels (adjusted OR [aOR] = 0.93, 95% confidence interval [CI]: 0.87–0.99, P = 0.048) and MA testicular histology (aOR = 30.98, 95% CI: 6.61–145.28, P < 0.001), as shown in Table 2. Other variables, including testicular volume and FSH levels, showed significant differences in univariable analysis but were not retained as independent predictors in the multivariable model.

Table 2.

Multivariable forward stepwise logistic regression of factors associated with successful sperm retrieval

Parameters	aOR (95% CI)	P
LH	0.93 (0.87 to 0.99)	0.048*
Histology (MA vs SCO)	30.98 (6.61 to 145.28)	<0.001*

^*Statistical significance at the P<0.05 level. LH: luteinizing hormone; MA: mixed atrophy; SCO: Sertoli cells-only; aOR: adjusted odds ratio; CI: confidence interval

Correlations with the number of frozen vials

Table 3 and Figure 1 present linear correlations between clinical parameters and the number of frozen sperm vials using Pearson correlation. Testicular size showed a significant positive correlation (right: r = 0.33, 95% CI: 0.12–0.52, P = 0.003; left: r = 0.33, 95% CI: 0.11–0.51, P = 0.004), while LH (r = −0.28, 95% CI: −0.48 to −0.05, P = 0.016) and FSH (r = −0.34, 95% CI: −0.52 to −0.12, P = 0.003) exhibited significant negative correlations. However, testosterone and prolactin were not significantly correlated (both P > 0.05).

Table 3.

Pearson's linear correlation between the number of frozen sperm straws and selected variables

Parameter	Pearson correlation coefficient (95% CI)	P
Right testicular size	0.33 (0.12 to 0.52)	0.003*
Left testicular size	0.33 (0.11 to 0.51)	0.004*
LH	−0.28 (−0.48 to − 0.05)	0.016*
FSH	−0.34 (−0.52 to − 0.12)	0.003*
Testosterone	0.04 (−0.19 to 0.27)	0.735
Prolactin	0.12 (−0.13 to 0.35)	0.354

^*Statistical significance at the P<0.05 level. FSH: follicle-stimulating hormone; LH: luteinizing hormone; CI: confidence interval

Figure 1.

Pearson’s linear correlation shows that testicular size, FSH levels and LH levels, correlate with the number of frozen vials in KS patients, whereas testosterone and prolactin levels do not. KS: Klinefelter syndrome; FSH: follicle-stimulating hormone; LH: luteinizing hormone.

Threshold values for predicting SR

To determine optimal thresholds distinguishing between positive- and negative-SR groups, we used recursive partitioning to create more homogeneous subsets based on different predictors. The differences between the subsets were evaluated using univariable logistic regression models. The optimal thresholds associated with a positive SR procedure for KS patients were LH level < 23.2 IU l⁻¹ (OR = 6.76, 95% CI: 1.41–32.49, P = 0.017), FSH level < 25.8 IU l⁻¹ (OR = 4.30, 95% CI: 1.54–12.05, P = 0.005), right testicular size ≥2.25 cm (OR = 4.32, 95% CI: 1.41–13.24, P = 0.011), and left testicular size ≥2 cm (OR = 2.95, 95% CI: 1.15–7.57, P = 0.035), as shown in Figure 2.

Figure 2.

Recursive partitioning of hormonal and testicular size thresholds differentiating between positive (blue) and negative (red) SR in KS patients. OR was calculated using univariable logistic regression model. SR: sperm retrieval; SRR: sperm retrieval rate; FSH: follicle-stimulating hormone; LH: luteinizing hormone; KS: Klinefelter syndrome; OR: odds ratio; CI: confidence interval.

Embryological and reproductive outcomes

Table 4 describes the embryological and reproductive outcomes among the subgroup of 18 positive-SR KS patients and their female partners that were treated in our medical center. A total of 46 ICSI cycles and 46 embryo transfer cycles were performed, with a total of 79 embryos transferred. The retrieved sperm achieved an average fertilization rate of 50.3%, and the resulting embryos achieved an average cleavage rate of 80.7% and an average blastulation rate of 40.2%. Regarding embryo quality, 45.1% on average of day 3 embryos were classified as good quality according to SART criteria for cleavage-stage embryos. Additionally, 55.0% on average of day 5 embryos were of good quality based on the Gardner blastocyst grading system. A total of 79 embryo transfers were performed, which resulted in an average clinical pregnancy rate of 39.1% and an average live birth rate of 39.5%.

Table 4.

Embryological and reproductive outcomes of Klinefelter syndrome patients with sperm retrieved during testicular sperm extraction/microtesticular sperm extraction procedure

Parameters	KS patients
Total patients, n	18
ICSI cycles, n	46
Total embryos transferred, n	79
Embryo transfer cycles, n	46
Fertilization rate (%), mean±s.d.	50.3±20.4
Cleavage rate (%), mean±s.d.	80.7±23.7
Good/fair day 3 embryos rate (%), mean±s.d.	45.1±27.7
Blastulation rate (%), mean±s.d.	40.2±35.8
Good/fair day 5 embryos rate (%), mean±s.d.	55.0±23.4
Implantation rate (per ETC; %), mean±s.d.	52.2±38.9
Clinical pregnancy rate (per ETC; %), mean±s.d.	39.1±28.6
Live birth rate (per ETC; %), mean±s.d.	39.5±28.4

KS: Klinefelter syndrome; ICSI: intracytoplasmic sperm injection; ETC: embryo transfer cycle; s.d.: standard deviation

Embryological and reproductive outcomes between TESE and micro-TESE procedures

Supplementary Table 2 presents a sub-analysis comparing embryological and reproductive outcomes between patients who underwent successful cTESE and those who underwent successful micro-TESE. While both groups demonstrated comparable fertilization, cleavage, and embryo quality rates, micro-TESE was associated with significantly improved clinical outcomes. Specifically, patients in the micro-TESE group had fewer embryos transferred on average (mean ± s.d.: 0.7 ± 1.0 vs 3.8 ± 4.4, P = 0.009) yet exhibited significantly higher clinical pregnancy (mean ± s.d.: 60.4% ± 27.8% vs 25.5% ± 20.6%, P = 0.017) and live birth rates (mean ± s.d.: 60.4% ± 27.7% vs 26.3% ± 20.3%, P = 0.018). No statistically significant differences were observed between the groups in implantation rates, blastulation rates, or the number of frozen straws (all P > 0.05).

Supplementary Table 2.

Embryological and reproductive outcomes by sperm retrieval technique (testicular sperm extraction vs microdissection testicular sperm extraction)

Parameters	TESE	mTESE	P
Total numbers, n (%)	18 (56.2)	14 (43.8)	-
Average number of frozen straws, mean±s.d.	10.0±6.1	8.0±5.0	0.320
Average total embryos transferred, mean±s.d.	3.8±4.4	0.7±1.0	0.009*
Average fertilization rate, mean±s.d.	47.6±19.2	56.4±24.8	0.501
Average cleavage rate, mean±s.d.	82.5±24.1	77.2±25.5	0.706
Average good/fair day 3 embryos rate, mean±s.d.	40.1±27.0	56.5±29.8	0.386
Average blastulation rate, mean±s.d.	34.6±34.7	55.0±42.7	0.512
Average good/fair day 5 embryos rate, mean±s.d.	55.0±28.5	55.0±70.7	1.000
Average implantation rate, mean±s.d.	42.5±41.7	67.6±30.9	0.192
Average clinical pregnancy rate, mean±s.d.	25.5±20.6	60.4±27.8	0.017*
Average live birth rate, mean±s.d.	26.3±20.3	60.4±27.7	0.018*

^*Statistical significance at the P<0.05 level. TESE: testicular sperm extraction; mTESE: microdissection testicular sperm extraction; s.d.: standard deviation

DISCUSSION

In this study, our goal was to refine the variables that influence SR success in both cTESE and micro-TESE surgical procedures in KS patients and to assess the subsequent fertilization potential for those patients who had viable sperm retrieved. By doing so, we aimed to optimize the clinical decision-making and enhance patient counseling regarding their reproductive potential.

The SRR in patients with KS varies across studies, ranging between 40% and 50%.11,12,19,20 A systematic review and meta-analysis reported an overall SRR of 44% per TESE cycle,11 and another study found a SRR of 45.4% in non-mosaic KS patients.12 Despite including a heterogeneous population of mixed ethnicities, our findings yielded a comparable SRR of 40.5%, making this the first study conducted in Israel on this topic. It is worth noting that this rate is comparable to non-KS NOA patients. A systematic review and meta-analysis reports an overall SRR of approximately 47% per TESE procedure,11 while other studies focusing specifically on idiopathic NOA (excluding KS and other genetic etiologies) report SRRs ranging from 20% to 42%.27,28,29

The mean age of KS patients undergoing these procedures is generally in the early 30s, as many cases of KS remain undiagnosed until couples encounter infertility challenges.11,13,19 This aligns with our findings, which showed a mean age of 32.2 years.

Younger age has been associated with higher SRRs. Studies, such as the one by Kang et al.,17 indicated that the SRR was significantly higher in the 20- to 29-year-old age cohort compared to those over 40 years of age. Renault et al.12 found no significant difference in SRRs between younger (15–22 years) and adult (23–43 years) cohorts, suggesting that delaying TESE does not substantially impair the SR outcomes. Similarly, in our study, we did not detect significant age-related differences between the positive and negative SR groups. This may reflect an underpowered sample or suggest that KS patients actually have a broader reproductive window than previously assumed. Further research is warranted to address this highly important topic.

cTESE and micro-TESE are established surgical options for KS patients seeking biological parenthood, but they are not without complications, such as hematoma formation, wound infection, devascularization, and hypogonadism.30,31 Therefore, careful patient selection is essential. Micro-TESE was developed in the late 1990s and first described by Schlegel in 1999. Micro-TESE was designed to maximize SRR while minimizing tissue excision.32 In our cohort, both cTESE and micro-TESE procedures were performed between January 2001 and June 2024, with earlier cases predominantly undergoing cTESE and later cases micro-TESE. Notably, 43.8% of patients in the positive SR group and 44.7% in the negative SR group underwent micro-TESE. While previous studies have shown that micro-TESE leads to higher SRR compared to cTESE,33 we were not able to corroborate these findings because short-term outcomes between cTESE and micro-TESE were comparable. This may be attributed to being underpowered. It is important to note that micro-TESE is gaining traction with all NOA patients. The American Society for Reproductive Medicine (ASRM) also acknowledges its superiority not only because of the occasionally reported improved results. It is also favored because it allows for taking smaller samples, thereby reducing the risks of long-term sequelae.30 Accordingly, we have moved to performing micro-TESE exclusively over the course of the years. Furthermore, our sub-analysis comparing reproductive outcomes between patients treated with micro-TESE and cTESE in patients with positive SRR showed that micro-TESE was associated with significantly higher clinical pregnancy and live birth rates compared to cTESE. This was despite no significant differences in fertilization, cleavage, or blastulation rates. Although fewer embryos were transferred in the micro-TESE group, outcomes were superior, potentially reflecting improved sperm quality or stricter embryo selection. Some studies and reviews note that micro-TESE may allow for more targeted sampling of seminiferous tubules, theoretically increasing the chance of retrieving more mature or motile sperm, but this has not been substantiated by direct comparative data on sperm quality endpoints.34,35

As mentioned, despite testicular atrophy and hyalinization, KS patients often retain focal areas of spermatogenesis, allowing for spermatozoa retrieval.9,10 Accordingly, in our study, patients with successful SR were more likely to exhibit MA histology rather than SCO. This suggests that failed retrieval may reflect more widespread testicular atrophy and hyalinization, where chances to sample areas with SCO are increased. These findings are in line with Almardawi et al.36 and Kang et al.,17 who showed that hypospermatogenesis (HS), which can also occur in men with MA, is associated with higher SRRs than SCO histology. Supporting this hypothesis, we also found that patients with successful retrieval had significantly larger median left and right testicular volumes, suggesting less testicular atrophy. Furthermore, testicular volume positively correlated with the number of frozen straws and recursive partitioning identified testicular size thresholds that were predictive of successful SR. These findings suggest that preserved testicular parenchyma may be associated with residual spermatogenic activity, aligning with current literature and supporting the inclusion of testicular volume in preoperative assessments.17,37,38

Hormonal assays are significant predictors of successful SR in TESE.12,15,39,40 AMH and inhibin B levels have been shown to be significant predictors of successful SR.12 We did not have these data; however, we found lower FSH and LH levels to be associated with successful SR, indicating less severe testicular failure, as stated in the previous literature.41,42 Furthermore, we established optimal thresholds values for LH, FSH, and testicular volume as predictors of SR success, providing potential clinical guidance for reproductive specialists. These thresholds may allow for more accurate patient preoperative consultation and set the appropriate expectations.

We were also able to show significant correlations of FSH and LH with the number of frozen sperm straws. This reaffirms their roles as not only qualitative but quantitative markers of testicular failure, a linear relationship which has not been demonstrated before. It is worth noting that while some studies have associated higher preoperative testosterone levels with increased SRRs,42,43 others have not. For example, Uemura et al.40 identified preoperative testosterone as a significant predictor of successful SR, with a cut-off value of 2.34 ng ml⁻¹. In our study, however, testosterone levels were not reliable predictors of positive SR and showed no correlation with the number of frozen straws. One possible explanation is the physiological distinction between free and bound testosterone: free testosterone refers to the unbound fraction circulating in the blood, whereas testosterone bound to proteins such as sex hormone-binding (SHBG) or albumin is biologically active and accessible to the testes.44 Another possible reason for this discrepancy is a study limitation. We lacked data on whether patients received preoperative testosterone replacement therapy (TRT), which could have influenced retrieval outcomes.41

Current literature investigating the embryological and reproductive outcomes of KS patients assisted by TESE-ICSI has shown compelling evidence. Fertilization rates in these patients are generally favorable, ranging from 52% to 63.5%.11,13,19,21 Our study, the first in Israel to assess embryo quality based on standardized grading criteria, demonstrated comparable results. Among the 18 KS patients analyzed, the fertilization rate was 50.3%, underscoring the viability of testicular sperm for ICSI. The cleavage rate was 80.7% and the blastulation rate reached 40.2%, both indicating good developmental potential. Notably, 45.1% of day 3 embryos and 55.0% of day 5 embryos were graded as good or fair using the SART criteria and Gardner grading system, respectively,25,26 suggesting a substantial proportion of embryos met quality thresholds associated with implantation.

Clinical and reproductive outcomes in the literature show greater variability. Implantation rates range from 26.2% to 34.8%,13,22 while clinical pregnancy rates span widely (from 39% to 70%, as shown in studies by Greco et al.23 and Schiff et al.45). Live birth rates also vary significantly, with Özkan et al.15 reporting 51.06% and Vloeberghs et al.22 noting 25.5% per embryo transfer cycle (ETC). In our study, a relatively high number of ICSI cycles and ETCs were performed compared to the existing literature.13,15,19,22,23 The relatively high embryo quality was reflected in an implantation rate of 52.2% per ETC, which compares favorably to rates reported in broader ICSI cohorts.46 The clinical pregnancy and live birth rates per ETC in our cohort were 39.1% and 39.5%, respectively. These findings highlight that testicular sperm from KS patients can not only fertilize oocytes but also lead to successful pregnancies and live births, a truly rare achievement.

While our study provides valuable insights into SR and reproductive outcomes in KS patients, several limitations must be acknowledged. First, as a single-center, retrospective study, the findings may not be fully generalizable to other populations. A multicenter or prospective study would offer a more comprehensive understanding of predictive factors for successful micro-TESE outcomes in KS patients. Second, due to the retrospective design and reliance on telephone questionnaires, there is a potential for recall bias. Third, as previously mentioned, we lacked data on whether patients received preoperative TRT. Fourth, although AMH and inhibin B levels are known predictors of successful SR, we did not have this information, which could make our predictive analysis less precise. Fifth and finally, the descriptive reporting of embryological and reproductive outcomes, along with the absence of detailed information on the female partner’s IVF protocols, limits our ability to draw definitive conclusions about their broader clinical significance.

CONCLUSIONS

This study provides a comprehensive evaluation of the factors influencing SR success and subsequent reproductive outcomes in men with KS undergoing cTESE or micro-TESE procedures. Our findings reaffirm that while conventional parameters such as age and procedural type may not independently predict SR outcomes, testicular volume and gonadotropin levels hold substantial prognostic value and present linear correlations with the amount of sperm retrieved. The identification of clinically actionable thresholds for LH, FSH, and testicular size enhances patient selection and surgical planning. Moreover, the embryological, morphological, and reproductive parameters observed in KS patients with successful SR were notably favorable, with fertilization, implantation, and live birth rates comparable to those of non-KS cohorts. Our study has several limitations due to its retrospective nature. These results support the consensus that genetic fatherhood is attainable for many KS infertile men and emphasize the reproductive viability of testicular sperm. However, these results should be reproduced in larger populations.

AUTHOR CONTRIBUTIONS

IA conceived the study design, collected the data, conducted the analysis, and drafted the manuscript. SB contributed to the study design and data interpretation and critically revised the manuscript for important intellectual content. YK and SEK provided expertise in laboratory and embryological aspects and contributed to data interpretation. FA and RH participated in clinical data acquisition and critical revision of the manuscript. SD contributed to the drafting and reviewing of the manuscript. NFW provided supervision, contributed to study conception and interpretation, and critically reviewed and edited the final manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.