In vitro effect of hCG on cryptorchid patients’ gubernacular cells: a predictive model for adjuvant personalized therapy

Abstract

Background

Cryptorchidism is the absence of one or both testicles in the scrotum at birth, being a risk factor for testis cancer and infertility. The most effective method to treat cryptorchidism is orchiopexy, followed by human chorionic gonadotropin (hCG) therapy; however, a portion of treated patients do not show a significant improvement in testis volume and vascularization after adjuvant therapy.

Methods

In this study, we generated an in vitro model to predict the patient response to hCG by cultivating and treating primary cells derived from five cryptorchid patients’ biopsies of gubernaculum testis, the ligament that connects the testicle to the scrotum. On these in vitro cultured cells, we analyzed the effect of hCG on cell proliferation, tubular structure formation, cellular respiration, reactive oxygen species content, and proteome.

Results

We demonstrate that in vitro hCG stimulates gubernacular cells to proliferate and form vessel-like structures to a different extent among the five cryptorchid patients’ cells, with a decrease in oxygen consumption and reactive oxygen species generation. Furthermore, from the proteomic analysis, we show that hCG regulates the intra- and extra-cellular organization of gubernacular cells together with a massive regulation of the antioxidant response.

Conclusions

Hereby, we characterized the cellular and molecular effects of hCG, demonstrating that the diverse patient response to hCG may be ascribable to their age since young patients better respond in vitro to the hormone, supporting a prompt surgical procedure and subsequent therapy.

Trial registration

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of “Azienda Ospedaliera Universitaria Integrata” (AOUI) of Verona, Italy (“ANDRO-PRO”, protocol code N. 4206 CESC of 26 April 2023).

Background

Testicular descent is a critical developmental event that ensures normal spermatogenesis. It requires the interaction of both anatomical and hormonal factors and can be divided into two phases: the transabdominal migration and the inguinoscrotal phase. The major hormone/receptor systems that are involved in the two phases of testis descent are, respectively, INSL3/RXFP2 (former LGR8) and testosterone/androgen receptor (AR) [1, 2]. The process is guided by two mesenteric ligaments: the cranial suspensory ligament (CSL) and the caudal genitoinguinal ligament (or gubernaculum testis). Testicles are attached to the diaphragm by the CSL that degenerates in males, allowing the testes to release and then migrate across the abdomen. Simultaneously, the gubernaculum goes through a thickening process, holding the testes very close to the developing internal inguinal ring, an event that causes their transabdominal migration into the inguinal region, followed by the inguinoscrotal phase during which the testes move from the inguinal region to the scrotum [3].

Cryptorchidism is defined as a medical condition characterized by the failure of one or both testes to descend from the abdominal cavity into the scrotal sac. It represents the most prevalent congenital anomaly observed in male infants, manifesting either as an isolated condition or concomitantly with additional congenital anomalies (syndromic cryptorchidism). Failure of the testes to normally descend can occur bilaterally in one-third of cases and unilaterally in two-thirds of cases, generally due to the failure of migration or to the failure of elongation of the spermatic cord postnatally [4]. However, also external factors, including maternal lifestyle and environmental pollutants, have been suggested to be included in the possible causes of cryptorchidism insurgence [5]. Retained testes are classified based on their location along the typical descent pathway (high/low abdominal, inguinal, suprascrotal, high scrotal) or classified as ectopic if located outside of this pathway. In clinical practice, however, a straightforward differentiation between palpable and non-palpable, as well as between unilateral and bilateral, is frequently employed [6]. Cryptorchidism, though generally regarded as a minor malformation, constitutes the most well-established risk factor for the onset of testicular cancer and infertility in adulthood [7]. Individuals with a historical occurrence of cryptorchidism frequently display sub-fertility arising from compromised spermatogenesis, predominantly observed in bilateral cases [8, 9].

Until few years ago, cryptorchidism was generally treated with a therapeutic approach that included only the use of human chorionic gonadotropin (hCG); nonetheless, this method was abandoned due to the low success rate (lower than 20%). Currently, orchiopexy—a surgical intervention—is favored in 95% of cases and demonstrates an approximate success rate nearing 100%. The surgery is conducted when the child is between 6 and 18 months old, as this period has a reduced likelihood of subsequent complications.

Indeed, our group reported that testicular volume and hormonal function at 18 years in patients diagnosed and treated for cryptorchidism during childhood are strongly conditioned by the age at which they underwent surgery and whether the undescended testis was unilateral or bilateral [3]. In addition to surgery, we have shown that post-operative hormonal treatment of patients with hCG improves vascularization, testicular volume, and morphology, supporting the adjuvant use of hCG therapy for cryptorchid patients to sustain their fertility potential [3]. Indeed, in this prospective study we demonstrated that, 6 months after orchiopexy, the achievement of a normal testicle size was evident in 81% of the subjects who also received adjuvant hCG therapy, compared to 46% of subjects who underwent only surgery [3]. In this scenario, it is noteworthy that a portion of patients treated with hCG still don’t respond to the therapy, without knowing whether the reasons for the non-response to hCG are due to the standardized fixed dosage or to the presence of molecular impairments in the signal cascade of hormonal stimulation. It is noteworthy that gubernaculum testis becomes rich in collagen and elastic tissue during testis descent, together with enrichment in vessels, suggesting that post-operative hCG stimulation may improve testicular growth by enhancing the vascularization of both the testes and gubernaculum [10, 11]. However, it has not been demonstrated whether these findings are also related to increased testicular function.

With this study, we aim to set the basis for developing an in vitro model that would predict the response to hCG post-surgical therapy through a personalized approach that includes in vitro culture and treatment of gubernacular cells derived from patients. The potential clinical application of our study will greatly impact the definition of proper dosage and type of treatment for each patient, avoiding a wrong therapy administration and, consequently, wasting time, which is stringently important in pediatric age.

Materials and methods

Patients

This study was approved by the Pediatric and Fertility Lab Internal Review Board and by the Institutional Ethics Committee of “Azienda Ospedaliera Universitaria Integrata” (AOUI) of Verona, Italy. Informed consent was secured from all parents and patients involved in the study. At our Institution, we collect samples of patients with primary azoospermia, genetic syndromes associated with azoospermia, undescended testis, testicular torsion, testicular trauma, and testicular neoplasms. We catalog each patient with a progressive identification number based on the surgery date. The tissue collection comprises biopsies of tissues obtained from 116 patients aged between 1 and 35. In this study, we considered as case studies those pediatric patients affected by undescended testes (see inclusion criteria) and as negative controls those patients who had already started or undergone pubertal development at the time of surgery and are affected by other andrological diseases than cryptorchidism.

Inclusion criteria

We considered all patients with undescended testes without any other genital malformations. None of the included patients had a personal or hereditary family history linked to the cryptorchid testicle. Patients treated by the same surgeon with the same surgical procedure (orchiopexy) were considered in this study. Also, patients with no previous hormonal therapy or previous inguinal surgical procedures were considered. All patients had testicular hypotrophy at surgery.

Concerning control individuals, we included patients affected by testicular pathologies different from cryptorchidism who are pubertal or adults. In particular, controls are affected by testicular torsion, who were clinically diagnosed and treated within 24 h of the onset of symptoms and surgically treated within 2 h subsequent to hospital admission, or by azoospermia, who underwent the testicular sperm extraction (TESE). All the patients (case studies and controls) enrolled in our study are reported in Table 1, along with their clinical presentation.

Table 1 Patients’ clinical details

Exclusion criteria

We did not consider as case studies those patients with non-palpable testes during the pre-operative visit and patients who required an intraabdominal approach.

Gubernaculum surgical collection

All patients underwent a standard inguinal surgery (orchiopexy); after surgical dissection, all testes were fixed into the scrotum. A gubernaculum testis biopsy (at least 5 mm from the lower pole of the testes) was collected without altering tissue organization or vascularization were performed.

Drugs and chemicals

Highly purified human chorionic gonadotropin (hCG) was extracted from pregnant women’s urine and was kindly provided by a producer company.

Tissue processing and in vitro culture

Gubernacula were processed as previously described by our group [12]. Briefly, we fragmented the piece of patient tissue with a scalpel in 500 µL of phosphate buffer solution (PBS) on a petri dish in sterility. Then, the tissues were enzymatically digested with type I collagenase and hyaluronidase at 37 °C for 1 h, repeating the incubation until achieving the complete homogenization of the tissues. Cell viability has been evaluated with Trypan blue assay. Finally, cells were maintained and grown in DMEM-Glutamax enriched with 10% fetal bovine serum (FBS) and 50 µg/mL gentamicin sulfate (all from Gibco/Life Technologies) and cultured at 37 °C with 5% CO₂.

Prior to hCG treatment, the cells were cultured for 16 h in the same medium but without FBS, which contains two gonadotropins (FSH and LH) at different concentrations depending on manufacturers’ lots. Then, cells were treated at the indicated doses and times in the medium without FBS. Bright-field cell images were acquired with an inverted microscope (Axio Vert. A1, Zeiss).

For the two adult control patients, we only had the availability of testicular biopsy. In this case, we processed the samples as described in Zampieri et al. [13]. Briefly, we disaggregated the testicular tissue by using an enzymatic mixture composed of DNAse, type I collagenase, and hyaluronidase, incubating for at least 1 h at 37 °C to obtain single cell suspension. Then, cells were grown in DMEM-Glutamax implemented with 10% FBS and 50 µg/mL gentamicin sulfate and subsequently incubated at 37 °C with 5% CO₂. The treatment with hCG was performed under the same conditions as gubernacular cells.

RNA extraction and qPCR

For cryptorchid and control patients, total RNA was extracted from 1 × 10⁵ cells using Single Cell RNA purification Kit (Norgen Biotek), in strict adherence to the manufacturer’s instructions. Whereas, for control cell lines (i.e., MCF7, Panc1, and fibroblast) total RNA was extracted from 1 × 10⁶ cells using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The integrity of RNA was assessed by electrophoresis on agarose gel. Subsequently, a total of 1 µg of RNA was employed for the synthesis of first-strand cDNA via RT-PCR. QPCR was performed in triplicate samples by SYBR-Green detection chemistry with GoTaq qPCR Master Mix (Promega, USA) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA).

The primers used in this study are reported in Table 2. The cycling conditions used were: 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s. The average cycle threshold of each triplicate was analyzed by the 2^−ΔΔCt method using SDHA as an endogenous control. Real-time PCR data were calculated relative to the positive control cells MCF7, a breast cancer cell line previously described in Dando et al. to express high levels of LHCGR [14], thus being considered as an internal positive control. Following, the reported fold change has been calculated over testicular cells derived from the control adult patient #40. Whereas for RXFP2 expression, the average cycle threshold of each triplicate was analyzed by the ΔCt method using SDHA as an endogenous control.

Table 2 List of primer sequences, Melting temperature (tm), and amplicon size, expressed in base pairs (bp)

Immunofluorescence analysis of LHCGR-positive cells

Gubernacular cells were fixed on Ibidi Imaging Chambers using 4% paraformaldehyde for a duration of 8 min, followed by three washes with PBS, each lasting 5 min. To block nonspecific binding sites, the cells underwent incubation at room temperature (RT) for 45 min in a blocking solution with 5% BSA in PBS. Afterwards, the samples were incubated overnight at 4 ◦C with an anti-LHR primary antibody (1:200; Thermo Fisher #8G9A2, Waltham, MA, USA) diluted in the blocking solution. After three washes with PBS of 10 min each, the cells were incubated for 1 h at RT in the dark with a specific secondary antibody (1 µg/mL) conjugated with Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA). The samples were mounted in an anti-bleaching medium (Dako Fluorescent Mounting Medium) and examined by confocal microscope (Leica TCS SP5 AOBS).

Cell proliferation assay

To analyze the cell proliferation rate, gubernacular cells were plated in 24-well cell culture plates (9 × 10⁴ cells/well, which correspond to T0) in DMEM-Glutamax supplemented with 10% FBS, 4.5 g/L glucose, and 50 µg/mL gentamicin sulfate (all from Gibco/Life Technologies, Waltham, MA, USA) and incubated at 37˚C with 5% CO₂. Viable cells were counted using Trypan Blue dye exclusion after 2, 4, 7, and 10 days of culture. The doubling time was calculated using the formula T = (T₂ − T₁) × log2/log (Q₂/Q₁), where: T₁, day 0; T₂, day 7; Q₁, cell number at day 0; and Q₂, cell number at day 7.

To test the effect of hCG on cell proliferation, gubernacular cells were seeded in the absence of FBS, as we previously described [14], to avoid interferences due to the presence of gonadotropins in this medium supplement. Cells were plated in 96-well cell culture plates (6 × 10³ cells/well) and incubated at 37˚C with 5% CO₂. Cells were treated with 100 IU/mL of hCG once a week for four weeks, and cell viability was measured at the end of the fifth week. We chose this treatment scheme to mimic the clinical posology as much as possible. Cell proliferation was assessed utilizing the crystal violet assay (Merck Millipore, Burlington, MA, USA) in accordance with the manufacturer’s protocol. Absorbance measurements were conducted through spectrophotometric analysis at 595 nm. The crystal violet assay is specifically designed for adherent cells, whereby non-viable cells detach from the plate surface. Consequently, this assay is deemed appropriate for in vitro investigations of cell proliferation and cytotoxicity, as corroborated by multiple manufacturer’s documentation.

Tubular structure formation assay

The formation of the tubular structure was analyzed by seeding gubernacular cells (1 × 10⁵ cells/well) above a Matrigel coating (50 µl/well) into a 96-well plate at 37 °C with 5% CO₂. To evaluate the effect of hCG on the formation of tubular structures, cells were suspended in an FBS-free medium complemented with 100 IU/mL of hCG. Five pictures per well (center of the well and four cardinal points) were taken every 3 h up to 12 h using Olympus Apexview Apx100. Two wells were seeded per conditions. Image analysis was performed using a specific program developed by ImageJ, an extension of the “Angiogenesis Analyzer”, a precise tool built into the ImageJ environment [15]. Heat maps of the numbers of junctions and of mean branching length (MBL) over time have been obtained using GraphPad Prism software (version 7.0). MBL values represent the mean of the lengths of trees composed of segments and branches in the analyzed area. Segments are identified as the pieces that join two junctions, while branches are the pieces starting from a junction branchpoint without joining other junction points.

Immunoblot analysis

Immunoblot assays were performed, as previously described [14], by analyzing whole lysate of gubernacular cells derived from patient #20 untreated (ctrl) or treated with 100 IU/mL hCG (the duration of the treatment is reported in each figure legend). Membranes were blocked in 5% low-fat milk in TBST (50 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20) for 1 h at RT and probed overnight at 4° C with anti-phospho (Ser133)-CREB (1:1,000; Invitrogen, Thermo Fisher Scientific, US #MA5-11192), anti-CREB (1:1,000; Invitrogen, Thermo Fisher Scientific, US #35–0900), and anti-TOM20 (1:1,500, Cell Signaling #42406). Horseradish-peroxidase-conjugated anti-mouse (1:10,000; KPL #074–1806) or anti-rabbit (1:10,000; KPL #074–1516) were used as secondary antibodies. The immunocomplexes were visualized by chemiluminescence using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the intensity of the chemiluminescence response was measured by processing the image using NIH ImageJ software (http://rsb.info.nih.gov/nih-image/). Amido black staining was used to confirm loading in different lanes.

Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) analysis

OCR and ECAR were measured in gubernacular cells derived from patient #20 by using a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Milan, Italy). Cells were seeded at the density of 1 × 10⁴ cells/well in a V7 XFe24-well cell-culture microplate and treated with hCG 100 IU/mL once a week for 4 weeks. The day of the analysis, cells were incubated in Mito Stress assay medium consisting of Seahorse XF DMEM Medium (Seahorse Bioscience, cat. No. 103575-100) in addition to 10 mM glucose, 0.5 mM Sodium Pyruvate, and 2 mM glutamine, pH 7.4, or alternatively in Glycolysis Stress assay medium consisting of Seahorse XF DMEM Medium (Seahorse Bioscience, cat. No. 103575-100) supplemented with 2 mM glutamine, pH 7.4, and incubated at 37 °C in a non-CO₂ incubator for 1 h. To perform the Mito Stress test, we recorded the OCR at the baseline and after sequentially adding 1 µM oligomycin A (port A), 3 µM of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, port B), and 0.5 µM each of Rotenone and Antimycin A (port C). The Glycolysis Stress test was performed by recording ECAR at the baseline and after sequentially adding 10 mM glucose, 1 µM oligomycin A and 50 mM of 2-deoxyglucose. OCR and ECAR raw data were normalized to the DNA content per well and quantified with the CyQUANT Cell proliferation assay kit (Thermo Fisher Scientific, cat. No. C35007) following the manufacturer’s instructions. Data related to mitochondrial respiration were calculated as described [16]. Data related to glycolysis were calculated as follows: Basal glycolysis: ECAR_BASAL—ECAR_2−DG; glycolytic capacity: ECAR_OLIGO—ECAR_2−DG; glycolytic reserve: ECAR_OLIGO—ECAR_BASAL.

Reactive oxygen species (ROS) levels analysis

The non-fluorescent diacetylated 2,7-dichlorofluorescein (DCF-DA) probe (Sigma-Aldrich), becoming highly fluorescent upon oxidation, was used to assess intracellular ROS production. Briefly, gubernacular cells derived from patient #20 were plated in 96-well plates (1 × 10⁴ cells/well) in 10% FBS medium. The day after, the culture medium was replaced with an FBS-free medium, and the cells were treated with hCG at 100 IU/mL. To analyze ROS levels produced daily for up to 7 days after hCG treatment, the cells were incubated in the culture medium with 10 µM DCF-DA for 15 min at 37 °C. The cells were rainsed with Hanks’ solution and the DCF fluorescence was measured using a multimode plate reader (Ex485 nm and Em535 nm) (Tecan Infinite 200 PRO). The values were normalized by Crystal Violet assay.

Pyruvate kinase activity assay

One µg of total protein extracts obtained from gubernacular cells, derived from patient #20 untreated or treated with 100 IU/mL of hCG once a week for 4 weeks were used to analyze pyruvate kinase (PK) activity, as previously reported [17]. Briefly, PK activity was measured according to published methods by a continuous assay coupled with lactate dehydrogenase (LDH). Absorbance change at 340 nm owing to oxidation of NADH was measured using Tecan Infinite 200 PRO. Kinetic assays for activity determinations contained recombinant pyruvate kinase (20–100 ng) or cell lysate (1–2 µg), Tris pH 7.5 (50 mM), KCl (100 mM), MgCl₂ (5 mM), ADP (0.6 mM), PEP (0.5 mM), NADH (180 µM), and LDH (80 units), Brij (0.015%), and DTT (1mM).

L-lactic acid quantification in culture medium

The culture media of gubernacular cells derived from patient #20 untreated or treated with 100 IU/mL of hCG once a week for 4 weeks have been retained and diluted 30-fold in H₂O. For each sample, 5 µL have been analyzed in a final reaction volume of 112 µL (Megazyme, #K-LATE 07/14). Absorbance at 340 nm was read after 10 min the reaction’s activation and L-lactic acid concentrations (g/L) were calculated according to the manufacturer’s instructions.

Proteomic analysis

Protein extraction and tryptic digestion

Gubernacular cells derived from patient #20 untreated or treated with 100IU/mL hCG once a week for 4 weeks were processed for protein extraction and peptide cleaned up using EasyPEP™ Mini MS Sample Prep Kit (Thermo Fisher). Extracted proteins were quantified by Pierce™ BCA Protein Assay Kit and 50 µg of protein sample was digested after incubation in a tryptic solution (furnished by Kit) with shaking at 37 °C for 3 h. Then, clean peptide samples were collected and quantified using Pierce Quantitative Fluorimetric Peptide Assay.

Mass spectrometry analysis

LC-ESI-MS/MS analysis was performed using an Ultimate 3000 nanoUPLC system (Thermo Fisher Scientific) coupled to Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). For reversed phase UPLC separation of peptides, 2 µL of solution (corresponding to 1 µg of the peptide mixture) were loaded onto the analytical column (Easy-Spray PepMap RSLC C18, 2 mm, 500 × 0.075 mm, Thermo Fisher Scientific) and separated applying a gradient from 4 to 50% acetonitrile (ACN) over 90 min. The eluant was ionized using a nanoESI source, operating in positive ion mode. MS1 spectra were acquired using the Orbitrap analyzer operating in data-dependent, ranging from 375 m/z to 1500 m/z, at a resolution of 120,000 (at 200 m/z), with standard automated gain control (AGC), and a maximum injection time of 50 ms. The MS2 spectra were acquired using the Orbitrap analyzer at a resolution of 50,000 (at 200 m/z). Precursor selection was based on their intensity from all signals with a charge state from 2 + to 5+, isolated in a 2.0 Da window, and fragmented in HCD mode using a dynamic exclusion of 60 s. For each condition, the experiments were performed in quadruplicate. LC-ESI-MS/MS data analysis was performed using Proteome Discoverer (v2.5). Identification was conducted using the Sequest HT search engine and the following parameters: stable modification carbamidomethyl (C), oxidation (M) and acetylation (protein N terminus) as variable modifications, the Uniprot database, trypsin as a specific protease, and a maximum of two missed cleavages. The “match between runs” option enabled the transfer of identifications across samples within 2 min of the aligned retention times. The confidence level for peptide identifications was evaluated by Percolator algorithm with decoy database searching. Identifications were filtered by FDR validation based on the q value; the strict FDR was set to 0.01 and the relaxed FDR to 0.05. Label-free quantification of identified proteins was referred to as unique peptides, requiring a minimum ratio count of two, and was calculated based on the raw spectral protein intensity. For each sample, raw intensities were logarithmized and normalized to the calculated average. Student’s t-test was performed on the normalized protein intensities. Proteins with p < 0.05 and a fold change > 1.3 were considered significantly altered in abundance between the samples.

Bioinformatic analysis

Functional enrichment assessment was conducted using ClueGO, a plug-in for Cytoscape (http://www.ici.upmc.fr/cluego/), alongside Gene Ontology (GO) annotation and protein-protein interaction network analysis via the STRING platform (http://string-db.org), as outlined previously [18]. In summary, the functional enrichment evaluation aimed to identify significantly enriched Reactome and KEGG pathways with a corrected p-value of less than 0.05. Furthermore, GO annotation utilized official gene symbols as identifiers and a Homo sapiens background, while the protein-protein interaction network analysis was executed at a medium confidence threshold (score 0.4), focusing exclusively on interactions that have been experimentally validated and sourced from a curated database.

Statistical analysis

Results are presented as mean +/- standard error of the mean (SEM) of three biological replicates at least. Statistical differences were determined by Student’s t-test two-sided and one-way analysis of variance (ANOVA) for multiple comparisons. Data analysis were performed by GraphPad Prism software (version 7.0) and statistical significance was defined as p < 0.05.

Results

In vitro culture of primary cells derived from cryptorchid and control patients

In this study, we aimed at the generation of an in vitro model to predict the response of cryptorchid patients to hCG therapy by exploiting the culture of a para-testicular biopsy, i.e., gubernaculum testis, which has been previously shown by our group to respond similarly to testicular cells to hormones in other andrological pathologies [12].

Firstly, we collected gubernacular biopsies from five cryptorchid patients (Table 1) who exhibited the inclusion criteria. Notably, this patient set covers a wide range of ages, from 1 to 10 years old. In addition to case study patients, we also analyzed four control cases. In particular, we obtained cells from gubernacular biopsies of two patients affected by testicular torsion, and from testicular biopsies of two azoospermic patients (Table 1). After tissue disaggregation, cells from all the patients grow in vitro, and the different cell types that constitute gubernaculum can be identified from a morphological point of view (Fig. 1A). Indeed, cells with the typical morphology of myofibroblasts/smooth muscle cells and fibroblasts are indicated with white and black arrows, respectively (Fig. 1A). Despite all cell samples being maintained in culture for at least three months, cells derived from the oldest cryptorchid patient (pt #94, 10 years old) showed a significantly lower proliferative capacity after 7 and 10 days in comparison to patients #7 and #20, who are 5 and 2 years old, respectively (Fig. 1B). Indeed, the doubling time for cells derived from patient #94 is about 4 days, whereas the values for cells derived from pt #7 and #20 are lower, i.e., 2.75 and 2.87 days, respectively, with a statistical difference among them (Fig. 1B). Interestingly, patient #94-derived cells present a significantly higher proliferation rate and a lower doubling time in comparison to the two controls analyzed, i.e., patients #40 and #116, indicating a slowed in vitro growth for adult-derived cells (Fig. 1B).

Fig. 1

Gubernacular cells derived from cryptorchid patients grow in vitro. A Representative bright field images of gubernacular cells derived from cryptorchid patients #2, #3, #7, #20, and #94, and of cells derived from control patients #15, #40, #55, and #116. Scale bar: 100 μm. B Proliferation rate of gubernacular cells derived from patients #2, #7, #20, and #94 (black symbols), and of cells derived from control patients #40 and #116 (white symbols). Values are reported as fold change relative to the beginning of the experiment (T0) and are the means (± SEM) of four independent biological replicates. Statistical legend: p < 0.5 (*) for pt #94 versus pt #7, #20, #40, and #116 after 7 days; p < 0.5 (*) for pt #94 versus pt #7, #20, #2, and #116 after 10 days. Based on these curves, we also report patients’ doubling time (days) and the significance relative to pt #94 derived cells

Gubernacular cells express receptors for gonadotropins and androgens

To better characterize in vitro cultured cells derived from cryptorchid and control patients we analyzed the mRNA expression levels of gonadotropin receptors, i.e., luteinizing hormone (LH)/choriogonadotropin receptor (LHCGR) and follicle-stimulating hormone receptor (FSHR), together with the androgen receptor (AR), which has been described to have a role in gubernaculum regression during the fetal period, regulating the testicular descent [19]. Regarding LHCGR, we analyzed the expression of the full-length receptor isoform at mRNA and protein levels in case study and control patients. Our data show that all the patients’ derived gubernacular cells express, to a different extent, mRNA levels of LHCGR full-length, as well as testicular cells from control patients (Fig. 2A). Noteworthy, despite LHCGR being a highly conserved gene among species, in humans and primates, its genomic organization has an additional exon, i.e., exon 6 A, that could be included through alternative splicing [20]. The resulting receptor variant lacks the transmembrane domain [21], giving rise to a soluble receptor that presumably presents a limited activation of the signal cascade [22]. Interestingly, real-time PCR analysis reveals a similar pattern of expression of the receptor short and long variants in all the patients (Fig. 2A). In addition, all patient-derived tissues express LHCGR full-length at the protein level, as observable in the immunofluorescent images in which the green spots correspond to the receptor (Fig. 2B). Additionally, our data show that FSHR is also expressed by all the gubernacular patients’ derived cells, as well as AR (Fig. 2A), confirming that not only testicular cells express gonadotropin receptors, as widely reported in the literature [23], but also gubernacular cells exhibit them, as we have previously shown for patients with testicular torsion [12]. These data lead us to exploit in vitro the effects of hCG treatment on gubernacular cells.

Fig. 2

Analysis of gonadotropin and androgen receptor expression in gubernacular cells cultured in vitro. A mRNA expression levels of LHCGR full-length, LHCGR short-length, FSHR, and AR in five cryptorchid patients’ derived cells (pt #2, #3, #7, #20, and #94) and three control patients (pt #15, #40, and #116). The mRNA levels of the receptors have been normalized on the housekeeping gene succinate dehydrogenase (SDHA) expression and reported as fold change relative to the levels of expression in testicular tissue derived from the control adult patient #40. Values are the means (± SEM) of four independent biological replicates. Statistical legend: (*) p < 0.05 versus pt #40. B Representative immunofluorescent LHCGR expression (green) images in gubernacular cells derived from cryptorchid patients (#2, #3, #7, #20, and #94), and from controls (#15, #40, and #116). Nuclei are marked with DAPI in blue and LHCGR long-variant in green. Scale bar: 50 μm. C RXFP2 expression values reported as the difference (D CT) with the SDHA housekeeping gene for each single sample. Values are the means (± SEM) of four independent biological replicates. n.d.: not determined, meaning that this sample does not express at any level the RXFP2 gene

Finally, it is noteworthy that mRNA expression of the receptor of INSL3, i.e., RXFP2, is expressed only by gubernacular cells, as reported in Fig. 2C, whereas testicular cells and three cell lines with different origins (such as foreskin, breast, and pancreas) do not express at any levels RXFP2, supporting the notion that it is a typical and specific gubernacular marker.

hCG stimulates cell proliferation and formation of tubular structures

To study and reproduce in vitro the effects of hCG treatment on the testicular volume enhancement evaluated in the clinic [3], we treated gubernacular cells derived from cryptorchid and control patients with hCG and then analyzed cell proliferation. We show that gubernacular cells derived from all the cryptorchid patients, except for pt #94, significantly respond to the hormonal stimulation compared to untreated cells derived from the same patient (represented as a dashed line in Fig. 3). Interestingly, after hCG treatment, cryptorchid patients show a significant increase in cell proliferation in correlation with age. Indeed, the two youngest case study patients, pt #20 and #3, present the highest response to hCG in comparison with the oldest cryptorchid patients, as well as versus controls (Fig. 3). An intermediate response is given by pt #7, who has an age that is halfway among the cryptorchid patients set. In line with this evidence, cells derived from the two oldest cryptorchid patients, i.e., pt #2 and pt#94, present a low stimulation or a non-significant regulation of cell proliferation by hCG treatment, with a statistically significant difference with the youngest ones. Finally, the two adult control patients (pt #116 and #40) do not respond to hCG, whereas the two other controls (pt #15 and #55), who are adolescents, present a slight increase of proliferation after hCG treatment of 132% and 118%, respectively. These data indicate that younger patients could respond better to hCG proliferative stimulation.

Fig. 3

Effect of hCG on gubernacular cell proliferation. The effect of 100 IU/mL hCG on cryptorchid and control patients’ cell proliferation has been evaluated by analyzing the data of hCG-treated cells (corresponding to the histograms) with those of untreated cells (dashed line), whose proliferation corresponds to 100%, for the same patient. White histograms represent control patients (#116, #40, #15, and #55), whereas grey histograms represent cryptorchid patients (#94, #2, #7, #20, and #3). Values are the means (± SEM) of four independent biological replicates. Statistical legend: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) for treated cells versus untreated cells of the same patient or, when indicated, among hCG-treated patient cells

The other aspect we evaluated in our in vitro model was the stimulation of tubular structure formation by hCG, a phenomenon comparable to the increased vascularization clinically demonstrated in patients treated with hCG [3]. Representative images of cells untreated and treated with hCG elaborated with dedicated software (as described in the methods section) are reported in Fig. 4A. Through this analysis, we compared the effect of hCG on all cryptorchid patients at four-time points (T1, T2, T3, and T4). As shown in Fig. 4B, we evaluated two parameters, such as the number of junctions and the mean branching length (MBL). The number of junctions indicates the points at which cells are effectively connected among each other through vessel-like structures (junctions are represented in blue surrounded by red in Fig. 4A). MBL represents the accurate measurement of branching and, therefore, provides a numerical description of the propensity of cells to create new connections with other cells (branches and segments are represented in green and yellow, respectively, in Fig. 4A). We evaluated these two parameters over the time after hCG treatment, in order to quantify the generation and the maintenance of the tubular structures. HCG-treated cells derived from the youngest cryptorchid patient (pt #3) present constant maintenance over the time of the number of junctions and MBL, indicating a strong capability to generate and conserve vessel-like structures (Fig. 4B). Oppositely, cells derived from the oldest cryptorchid patient (pt #94) show a progressive decrease of junctions and MBL over time, supporting a low vasculogenic propensity of these cells after hCG treatment. The other cryptorchid patients, i.e., pt #7 and #20, aged between pt#3 and #94, present an intermediate capacity to form junctions, whereas they show a consistent capacity to maintain MBL over time with a pattern similar to that of patient #3 (Fig. 4B). Control patients, on the other side, present a diminished capacity to form junctions over time to a similar extent to patients #2 and #7; regarding MBL, controls clearly lose the capacity to conserve these tubular structures with a pattern similar to the oldest patients, in an exception for control patient #15. These data are further confirmed by the quantitative analysis of the number of junctions and MBL after 12 h of hCG treatment (corresponding to T4) relative to T0 (Fig. 4C). Taken together, these data indicate that cells derived from younger patients present a more marked ability to generate and maintain tubular structures, correlating with vasculogenic ability, under hCG stimuli compared to oldest patients and controls.

Fig. 4

Effect of hCG on tubular structure formation of gubernacular cells. A Representative images of tubular structures analyzed with the dedicated software. Magnification: 4X. B Heat-map representation of the number of junctions and the main of branching length (MBL) of hCG-treated gubernacular cells derived from cryptorchid patients (#94, #2, #7, #20, and #3) and control patients (#116, #40, and #15) at four different time points: after 3 (T1), 6 (T2), 9 (T3), and 12 (T4) hours from the seeding. C Quantitative representation of the number of junctions and MBL at T4, reported as fold change relative to T0 (dashed line). Statistical legend: (*) p < 0.05 for T4 versus T0 for each patient’s derived cells

hCG decreases the oxygen consumption rate and the reactive oxygen species content in gubernacular cells

The induction of cell proliferation by hCG led us to investigate whether this hormone could alter the energetic metabolism of cells. For this aim, we chose a representative patient, i.e., patient #20, since his cells well respond to hCG stimulation in vitro and, from a technical point of view, the relative cell proliferation rate is compliant. Before analyzing the metabolic regulation of these cells by hCG, we confirmed that this hormone activates the signaling cascade downstream of LHCGR by analyzing the phosphorylation of the transcriptional factor CREB, a known target subsequent to LHCGR activation [24]. Indeed, after 10, 20, and 30 min of treatment of gubernacular cells with hCG, there is a significant increase of phospho-CREB compared to untreated cells (Fig. 5A), corroborating the binding of hCG to LHCGR and the activation of the signal cascade. Hence, we performed the analysis of oxygen consumption rate (OCR - Fig. 5B) and extracellular acidification rate (ECAR - Supplementary Fig. 1A) of patient #20 cells untreated (control) and treated with hCG. Our data on OCR results show that the basal and maximal respirations are significantly decreased after the hCG effect, along with low levels of spare respiratory capacity and ATP-driven respiration (Fig. 5B). These data suggest that, after hCG treatment, gubernacular cell proliferation is connected with a decreased respiratory rate of the cells. Surprisingly, the decreased oxidative metabolism is not associated with a concomitant glycolysis (ECAR) increase after hCG exposition, as reported by our data in Supplementary Fig. 1A. To further corroborate that hCG does not exert an effect on glycolysis, we analyzed the activity of pyruvate kinase (PK) (Supplementary Fig. 1B), the expression levels of lactate dehydrogenase (LDH), and the level of lactate secreted in the cell medium (Supplementary Fig. 1C). Our data demonstrate that hCG treatment does not significantly alter these three parameters, supporting the evidence that the hormone does not target glycolysis in vitro. In particular, regarding LDH, we extrapolated the expression levels of the two key subunits of the enzyme, i.e., LDHA and LDHB, from the untargeted proteomic analysis. This analysis shows that both subunits’ expression levels are not modulated in treated cells compared to control cells, with a fold change of 0.914 and 0.905, respectively. Finally, to exclude that the effect of hCG on the metabolism could be due to an adaptation of cells to the long exposure (i.e., 4 weeks), we also analyzed OCR and ECAR after 1 week of hCG treatment, and a not significant modulation has been detected (data not shown).

Fig. 5

hCG treatment impairs oxygen consumption parameters and decreases ROS levels. A Representative immunoblots of phospho- and total-CREB normalized on Amido Black in gubernacular cells derived from patient #20 treated with 100 IU/mL hCG for 10, 20, or 30 minutes compared to untreated (ctrl) cells. The densitometric analysis is the average of three biological replicates. Statistical legend: p < 0.05 (*) for hCG-treated cells versus untreated cells, and p < 0.05 (^#) for treated cells relative to 20’. B Oxygen Consumption Rate of gubernacular cells derived from pt #20 untreated (control) and treated with 100 IU/mL hCG. Histograms legend: white: untreated (ctrl) cells; grey: hCG-treated (hCG) cells; extrapolated data include basal, maximal respiration, spare respiratory capacity and ATP-driven respiration. C Analysis of intracellular ROS production on gubernacular cells derived from pt #20 untreated (white histogram) and treated with hCG (grey histogram). ROS levels were analyzed every day up to 7 days after hCG treatment. Values are presented as mean ± SEM of three independent biological replicates. Statistical legend: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) for hCG-treated cells versus untreated cells. D Representative immunoblots of the mitochondrial protein TOM20 normalized on Amido Black in gubernacular cells derived from patient #20 treated with 100 IU/mL hCG once a week for 4 weeks compared to untreated (ctrl) cells. The densitometric analysis is the average of three biological replicates

To investigate the molecular effect of hCG, we also analyzed the reactive oxygen species (ROS) levels through a fluorescent assay to further confirm that hCG affects oxidative phosphorylation. Indeed, it is known that mitochondria generate approximately 90% of cellular ROS, which is strictly connected with alterations of electron transport complex activity [25]. Based on this concept, we show that hCG significantly decreases ROS levels compared to control cells and maintains this antioxidant effect after 7 days from the hormone exposition (Fig. 5C). Finally, to exclude that the effect of hCG on mitochondrial metabolism is due to a decrease in mitochondrial mass, we analyzed the expression of TOM20, a known protein of the outer mitochondrial membrane whose expression correlates with mitochondrial mass. As reported in Fig. 5D, TOM20 expression levels are not significantly modulated in the gubernacular cells of patient #20 treated with the hormone, further corroborating that the hormonal effect is directly linked with a decrease in oxygen respiration and ROS production.

Proteomics reveals remodeling of intracellular organization and extracellular matrix composition by hCG treatment

To obtain information about the modulation induced in vitro by hCG at the protein level, untreated and treated gubernacular cells derived from patient #20 were subjected to proteomic analysis. By high-resolution accurate-mass Orbitrap MS, we identified 3,776 proteins (Additional file 2), and differentially expressed proteins (DEPs) were detected between the untreated and treated cells. Among these DEPs, 27 were significantly up-regulated, and 69 were significantly down-regulated after the hCG treatment. A Cytoscape analysis was performed to investigate the main pathways involved in hCG treatment (Fig. 6A and Additional file 3). Overall, the DEPs showed a significant link with different Reactome and KEGG pathways, including, among the most interesting, extracellular matrix (ECM) organization, formation of elastic fibers and associated molecules, reorganization of the ECM composition (such as integrins, glycosaminoglycans, laminins, and proteoglycans), and differentiation processes linked with developmental cell lineages. All these data support the physiologic role of hCG during gestation in stimulating gubernaculum shaping to support testis descent in the scrotum. This evidence is further supported by the Gene Ontology (GO) enrichment analysis (Fig. 6B and Additional file 3), where it emerged that four categories of the molecular function (MF) branch are linked with ECM composition, structural constituents, and molecules. In addition, the cellular component (CC) and biological process (BP) branches highlight six categories connected with the extracellular matrix and, interestingly, two categories connected with elastic fiber assembly and collagen production (Fig. 6B). This analysis further supports the role of hCG during the gubernaculum re-shape process for testis descent.

Fig. 6

Bioinformatic results of proteomics. A Visualization of Reactome and KEGG pathways with Cytoscape characterizing the de-regulated proteins (DEPs) in cells derived from pt #20 treated in vitro with hCG once a week for 4 weeks, versus control cells derived from the same patient. Hexagons and rectangles represent KEGG and Reactome pathways, respectively. The node size is proportional to the number of proteins, and the node color depicts the enrichment significance (ranging from red = p-value < 0.05 to dark red = p-value < 0.005 and dark brown = p-value < 0.0005). B GO enrichment analysis of DEPs retrieved using String. The six most significantly (p < 0.05) enriched GO terms in molecular function (MF), cellular component (CC), and biological process (BP) branches are presented. C STRING-based interaction analysis of DEPs. The circles represent the identified proteins, and the edges represent protein–protein interactions. Blue lines represent known interactions from curated databases, pink lines represent experimentally determined interactions, purple lines indicate that protein homologs are found interacting in other organisms

Additionally, three other interesting pathways, which emerged by the bioinformatic analysis as being implicated by the hCG treatment (Fig. 6A), are: the TGF-b and IGF signaling, both involved in the functionality of the testis [26, 27]; the NFE2L2 and cysteine/methionine that are reported to be strictly connected with oxidative stress [28, 29]; and finally the TP53 dependent transcription of DNA repair genes.

Moreover, the network interaction between DEPs was analyzed using the STRING database. As shown in Fig. 6C, the protein-protein interaction (PPI) network derived from DEPs in the hCG-treated cells exhibited a medium average node degree as indicated by three sub-networks of interacting proteins. In particular, these sub-networks included: interacting proteins (in red) strictly associated with extra-cellular matrix composition, including FBLN2, FBN1, NID2, TGFBI, TNC, and VCAN; interacting proteins (in pink) that regulate steroids synthesis and GTPase mediated signal transduction regulation, such as ARL3, HSD3B7, and SQLE; and interacting proteins (in green) related to the regulation of transcription, thus supporting a modification of cellular proliferation, including CDK9, GTF2H1, HNRNPR, and SUP5H.

Finally, to investigate the antioxidative effect of hCG further, we listed the DEPs linked with oxidative stress in Table 3. Among these, it emerged that eight proteins are down-regulated in hCG-treated cells, such as: 1) exonuclease 3’−5’ domain-containing protein 2 (EXD2), the reduced levels of which increase ROS production [30]; 2) glutamate-cysteine ligase regulatory subunit (GCLM), which is the rate-limiting enzyme of glutathione synthesis and is involved in the antioxidant response [31]; 3) cytoplasmic aconitate hydratase (ACO1), which is strictly connected with the iron-regulatory protein IRP1 [32], a protein that coordinates the cellular iron metabolism and that also functions as a ROS sensor [33]; 4) thioredoxin (TXN), 5) thioredoxin domain-containing protein 17 (TXNDC17) and 6) sulforedoxin-1 (SRXN1), which are known protectors against reactive oxygen stress [34, 35]; 7) bis(5’-nucleosyl)-tetraphosphatase (NUDT2), which belongs to the nudix pyrophosphatase protein family that protects cells from oxidative stress [36]; and 8) metalloreductase STEAP3 (STEAP3), whose knockdown has been shown to significantly increase ROS levels in renal cell carcinoma [37]. On the other side, two proteins involved in the regulation of oxidative stress are up-regulated, such as: (1) decapping enzyme scavenger (DCPS), which has been shown to sustain 5 different pathways involved in oxidation-reduction processes, especially GLRX, the reduced glutathione (GSH) oxidoreductase that is a ROS scavenger [38]; (2) the receptor-type tyrosine-protein phosphatase (PTPRS) has a role in oxidative stress mechanism indeed, its overexpressing increases ROS production and promotes apoptosis through TLR4/NF-κB pathway [39]. The massive regulation of the expression of proteins involved in the antioxidant response after hCG exposition supports that this hormone decreases total intracellular ROS levels and, consequently, cells decrease their antioxidant armory. In conclusion, we can assume that hCG targets oxidative phosphorylation together with a conceivable slackening of the electron transport chain could cause a decrease in ROS levels.

Table 3 List and abundance ratio (hCG-treated cells versus control cells) of up- and down-regulated proteins involved in the regulation of the oxidative metabolism (p < 0.05). Up arrows and down arrows indicate up- and down-regulation, respectively

Discussion

As previously reported by our group, cryptorchid patients who underwent orchiopexy followed by subcutaneous administration of hCG show a significant increase in testicular volume and vascularization compared to untreated patients [3]. However, despite this important evidence about the effect of hCG, a portion of patients (about 40%) still does not respond to the therapy. Up to now, it is unknown whether the reason for the non-response to hCG is linked to the unique posology given to different patient subtypes or the presence of alterations in the signal cascade of this hormonal stimulation. With this study, we aimed to perform the first step towards the scenario of precision medicine by creating the basis for generating a personalized and predictive model of therapeutic response for cryptorchid patients after orchiopexy. For this aim, we cultured in vitro primary tissue biopsies derived from the para-testicular component, gubernaculum testis, of cryptorchid patients who underwent surgery. Gubernaculum testis is histologically composed of an abundant extracellular matrix rich in glycosaminoglycans and mesenchymal cells, including fibroblasts and smooth muscle cells. We recently demonstrated that, after its mechanical and enzymatic disaggregation with a protocol optimized by our group, it grows in culture and, overall, it resembles the testis’ behavior [12], by sharing with it many properties. In addition, from a clinical point of view, since cryptorchid patients generally exhibit a retained small testicle, which could present alterations in adult age (including fertility problems, hypogonadism, and tumor development [40]), the biopsy and study of gubernaculum lead to maintain completely intact the testicle.

In this study, we considered five pediatric patients who respected the inclusion criteria. It is noteworthy that also patient #7, who presents a retractile testicle, has been included since this condition is considered a variant of cryptorchidism in terms of surgical procedure, low testis volume, non-completely regression of gubernaculum testis, and long-term effects on the fertility potential. Although orchiopexy is generally performed between 6 and 18 months of age, the age of cryptorchid patients enrolled in our study is heterogenous, with two of them that are 1 and 2 years old (pt #3 and #20, respectively), one who is middle-aged being 5 years old (pt #7), and two that are advanced-aged patients being 8 and 10 years old (pt #2 and #94, respectively). Despite a common in vitro behavior of all the patients’ derived cells, in terms of maintenance in culture for over 3 months and the composition of mixed cellular populations, the proliferation rate was different among them. Indeed, cells derived from the oldest cryptorchid patient (pt #94) present a slower proliferation rate and a lower doubling time compared to patients #20 and #7, who are younger than patient #94. For this assay, we were not able to analyze the proliferation rate of cells derived from patient #3 due to their senescence triggering.

Then, we demonstrated that gubernacular cells share some similarities with testis, including the expression of gonadotropin receptors (LHCGR and FSHR), thus representing a good candidate to study the response of testicular cells to hCG stimulation. Specifically, in gubernacular cells, the presence of the short variant of LHCGR, which includes the exon 6 A, is intriguing due to its presumably soluble form; however, this evidence requires more strengthening by the literature, since it may open the way to future consideration of LHCGR short variant expression levels in the prediction of the patient response to hormonal therapy. On the other side, as expected, the androgen receptor is also expressed by gubernacular cells since, during fetal life, it responds to the stimulation of testosterone to regress and favor the testicular descent into the scrotum [19]. Indeed, different studies showed that testosterone could be produced during infancy, around the first year of life, as a residual secretion from pregnancy [41].

The effect of hCG on the proliferation of gubernacular cells derived from cryptorchid patients shows that they respond in vitro to the therapy by increasing their proliferation. Interestingly, the strength of the effect is higher in younger patients than in the oldest one. To further support this evidence, cells derived from control patients, who are pubertal or adult, show a significantly lesser extent of cell proliferation in comparison to younger cryptorchid patients after hCG treatment. Finally, also the analysis of the capacity of treated gubernacular cells to form and maintain during the time tubular structures, which resemble a vessel-like organization, supports the diverse response among cryptorchid patients of different ages. This result is noteworthy since the gubernaculum contributes in supplying the blood to the testicle [42]: here, we suggest that hCG treatment could improve the testicle functionality also by supporting the gubernaculum and thus, from a clinical point of view, during orchiopexy, it is recommendable to maintain the cranial part of the gubernaculum that may offer more functional support. In fact, after surgery, when the testis is fixed into the scrotum, the residual part of the gubernaculum may create a new vascularization web with the dissected scrotum. Interestingly, a difference among control patients emerged regarding MBL evaluation, specifically for patient #15, who is affected by testis torsion. Indeed, the tissue derived from this patient is gubernaculum testis, unlike the other two controls, demonstrating that the different effect of hCG on the generation of vessel-like structures in the control group is due to the origin of the tissue biopsy.

The second part of this study aimed to investigate the metabolic alterations induced by hCG. Our results indicate that the hormone partially inhibits oxygen consumption, suggesting that oxidative metabolism is slowed by hCG treatment, without altering the mitochondrial mass. On the other side, the analysis of glycolysis shows that this metabolic pathway is not affected after hCG exposition, as confirmed by ECAR assay and analysis of pyruvate kinase activity and lactate secretion. Accordingly, a previous study performed on Leydig cells derived from mice showed that the stimulation of LHCGR increases the dependence on glycolysis and decreases the dependence on oxidative metabolism [43]. Nevertheless, to completely understand the metabolic arrangement of hCG-stimulated cells, future analyses are needed, for instance, a metabolomic approach, to understand how cells compensate for the alterations of the energetic metabolism and produce the ATP necessary to sustain the increased cellular proliferation by hCG. In line with the decreased oxygen consumption, we show that hCG drops the levels of ROS together with a regulated expression of many antioxidant enzymes or enzymes involved in the regulation of oxidative stress response. This finding suggests that hCG decreases cellular respiration by slowing the electron transport chain within a threshold tolerable by the cells. Furthermore, it is important to note that hCG’s in vitro antioxidant action is maintained for 7 days after the treatment. At this time, the next treatment is administered, allowing the inference that the oxidative state can be kept low for the duration of therapy. Finally, the untargeted proteomic analysis led us to investigate the molecular actions of hCG and, in particular, our data further evidence the role of the hormone in the remodeling of gubernaculum at intra- and extra-cellular levels, in line with the role of hCG during pregnancy and testicular descent.

Future studies are needed to validate the use of gubernaculum as an in vitro model to predict the response to hCG therapy in a personalized way, in particular by (i) implementing the number of patients, (ii) performing a parallel study on the patient and on the cells derived from the same patient between our in vitro model and the clinical evaluation of hCG effects, (iii) testing a range of hCG doses, and (iv) analyzing the effects of other hormones, including the follicle-stimulating hormone (FSH).

Conclusion

In this study, we propose a personalized in vitro predictive model of hCG response for cryptorchid patients after orchiopexy by exploiting a small gubernacular biopsy taken during surgery. The evidence of this study is also applicable to the surgical practice since we show that maintaining intact the gubernaculum is crucial to support testicular functionality. Indeed, the salvage of the gubernaculum, or at least its cranial part, near the lower pole of the testes, may be associated with improvement of the testicular volume, function, and better response to hormonal therapy. In our study, we also highlighted the possibility of directly analyzing the levels of hormones’ receptors by extracting RNA from patient biopsies, thus limiting the culture sample size, to perform a pre-screening to identify candidates to be investigated in vitro. Conclusively, our in vitro system represents a predictive model of therapy response in the pre-clinical phase, revealing that, to achieve a better hCG effect as adjuvant therapy after orchiopexy, it would be crucial to treat patients as early as possible, since a stronger in vitro effect is evident in younger patients.