Study design
This prospective cohort study was conducted at the IbnSina IVF Centre, Sohag, Egypt, in collaboration with the Zoology Department, Faculty of Science, Assiut University, and the Dermatology & Andrology Department, Faculty of Medicine, Assiut University. The study was prospectively approved by the Ethics Review Board of Assiut University (no. 17100808; 18 August 2019). Eligible participants gave written informed consent for participation after receiving detailed explanations on the study. The study had no interventions or assignment to any study arms.
Study participants
Men with non-obstructive azoospermia were eligible if they were aged between 25 and 55 and were undergoing a mTESE procedure. They were invited to participate in the study if they had a clinical diagnosis of non-obstructive azoospermia. The exclusion criteria were presence of a varicocele, exposure to reproductive toxicants, or having a systemic disease known to affect spermatogenesis. When sperm were identified in the testicular tissues, they were retrieved for use in an ICSI cycle. Women were eligible provided they were 18 to 40 years of age and were undergoing an ICSI attempt. Causes of infertility were male factor only or combined with female factor. Surgically retrieved sperm, either fresh or frozen-thawed, were suitable for the study. Patients who refused to take part in the study were deemed ineligible. Women with thin endometrium (≤ 7 mm), uterine anomalies, untreated hydrosalpinx or a severe medical condition were accounted for during the study.
Initial evaluation of male patients
A clinical history was taken for all eligible men. A genital and scrotal ultrasound examination was performed to measure testicular size and rule out any pathologies. Men were diagnosed as being azoospermic if no spermatozoa were found in three conventional semen analyses that included centrifugation and examination of the pellet according to the WHO protocol (WHO 2010). Each man’s serum levels of follicular stimulating hormone (FSH) and testosterone were determined by automated immunoassay assay (MINI VIDAS®, Biomerieux, France).
mTESE procedure
All trials for sperm extraction were performed under surgical microscopy by microsurgical testicular sperm extraction (mTESE) as described earlier [14]. Briefly, under surgical microscopy (MS-1600, Medical Microsurgery, Medical sources Limited Co.) and local anesthesia, we began with the testis that had the larger volume, otherwise the right testis. The testis was visualized via a scrotal midline incision, followed by transverse incision of the tunica vaginalis. Using a magnification power of × 20–50, the thicker and more opaque seminiferous tubules were identified and biopsied for determination of sperm presence.
In a 30-mm Petri dish (Falcon, BD, USA), testicular tissues were washed in 3 mL Global Total HEPES medium (LifeGlobal, CooperSurgical, USA), then transferred to a clean dish containing 3 mL Global Total HEPES medium and dissected with small scissors, followed by vigorous mechanical milking and mincing of the tissue with two needles of insulin syringes. The dish contents were then examined under an inverted microscope (× 40, Olympus IX 71, Japan) at room temperature. The surgical procedure was halted as soon as sperm were detected. If sperm were not detected, further biopsies from a different area of the testis, and from the other testis underwent the same preparation and examination procedures.
The sperm-positive biopsies were minced, and the sperm suspension was separated from the tissue remnants using a glass Pasteur pipette. The sperm suspension was then diluted with Sperm CryoProtec medium (Nidacon, Mölndal, Sweden) in a 2:1 ratio before being cryopreserved in cryovials according to the manufacturer’s protocol. A small fraction of the cryopreserved processed testicular tissue from each biopsy was retained for later gene expression analysis [15], while the rest of the sperm remained frozen until they were thawed for use in an ICSI cycle.
Part of the testicular tissue was fixed in Bouin’s solution for histopathological examination in all patients.
Testicular gene expression analysis
To isolate the RNA, all tissue samples from each patient were treated with Qiazol lysis reagent with RNase inhibitor (Qiagen, Germany). The redissolved RNA in RNase free water was then cleaned using a miRNeasy Mini kit (Qiagen, Germany) to remove all contaminants, inhibitors, and residuals before processing. This step was followed by cDNA synthesis for each extracted sample by high-capacity cDNA reverse Transcription with RNase inhibitor kit (Applied Biosystems, UK)
Expression level of the MND1 target gene was measured using SYBR Green-based real-time PCR, and consequent relative quantification analysis with the aid of GAPDH as a housekeeping gene on the Step-One real time PCR system (Applied Biosystems, UK). The 20 μL PCR reaction mixture for the target gene and the corresponding housekeeping gene for each sample included 15 μL master mix with 9 μL PCR-grade water, 1 μL forward primer for each parameter and the housekeeping gene (20 pmol/μL), 1 μL reverse primer for each parameter and the housekeeping gene (20 pmol/μL), 4 μL ready to use SYBR Green universal master mix (Applied Biosystems, UK), and 50 ng cDNA. The thermal profile was as follows: initial denaturation at 95 °C for 10 min, followed by 45 cycles of amplification, starting with denaturation at 95 °C for 10 s, annealing at 66 °C for 20 s and extension at 72 °C for 25 s. Following amplification, an extra cycle of melting curve analysis was done for product characterization by heating the reaction mixture from 65 °C to 95 °C at a rate of 0.2 °C/s. The Step-One real time PCR system software automatically calculated the gene expression values by relative quantitative analysis.
ICSI protocol
Stimulation cycles were controlled with either GnRH agonist (Decapeptyl 0.1 mg, Ferring) started on day 21 of the preceding cycle, or GnRH antagonist (Cetrotide® 0.25 mg, Merck Serono) started on stimulation cycle day 6. For ovarian stimulation, patients received 150–300 IU rFSH (Gonal-F, Merck Serono) and hMG (Menogon, Ferring) in a 2:1 ratio, with the dose adjusted according to the response. Oocyte retrieval took place 37 h after the GnRh agonist (Decapeptyl 0.1 mg, Ferring) or hCG (500 g, Ovitrelle, Merck Serono) maturation trigger was given. Follicles were aspirated into 14 ml tubes containing 1 ml Global HEPES Total medium (LifeGlobal, USA), held in a 37 °C tube warmer (IVFtech, Denmark).
Oocytes were denuded 39 h after the hCG trigger, in Global Total HEPES medium (LifeGlobal) containing 40 IU hyaluronidase (Irvine, USA), using a 170-μm denuding pipette (Cook, USA). Oocyte denudation was performed at 37 °C. All MII oocytes underwent ICSI in Global Total HEPES (LifeGlobal) immediately post denudation.
All cryopreserved testicular samples were thawed as per our standard protocol [16]. Briefly, samples were removed from cryostorage and incubated in a 37 °C water bath for 20 min, followed by centrifugation and washing twice in PureSperm Wash media (Nidacon, Mölndal, Sweden) at room temperature. The washed pellet was suspended in 0.5 mL PureSperm Wash supplemented with 2 mg/mL pentoxifylline 15 min before use. Sperm were then washed to remove the pentoxifylline (Sigma) before use in ICSI. Search dishes for sperm contained 5 × 20 μl droplets surrounding 2 × 2 μl PVP droplets. We chose motile sperm (if available) with normal morphology, or as close to normal morphology as possible, for ICSI using an inverted microscope (40X lens, IX 71, Olympus, Japan). As per our standard protocol, using a Sunlight injection pipette (Sunlight, USA), we deposited each immobilized spermatozoon into the cytoplasm after mechanical piercing of the zona pellucida and cytoplasmic membrane [17].
From days 0 to 5 or 6 after injection, all injected oocytes were cultured in Global Total medium (LifeGlobal) in a microdroplet dish (Vitrolife), three per 20 μl droplet, overlaid with 5 ml oil (NidOil, Nidacon, Mölndal, Sweden), without medium renewal. A fertilization check and embryo grading were performed on days 1, 3, and 5 of culture as per the Istanbul Consensus [18]. Oocyte and embryo culture was performed at 37 ± 0.1 °C in a Minc-1000 incubator (Cook, USA) supplied with premixed gas (7.5% CO2, 5% O2 and 87.5% N2) to achieve pH 7.25 ± 0.02. One to two blastocysts were transferred to each woman under ultrasound guidance using a Sydney IVF embryo transfer catheter (Cook, USA). All women received intramuscular progesterone (100 mg, Prontogest, IBSA) started from the day after oocyte retrieval until the 12th week of gestation or a negative pregnancy test.
Outcomes
The non-obstructive azoospermia is defined as the absence of spermatozoa in the ejaculate due to lack of production of mature spermatozoa. The primary endpoint of this study was presence or absence of sperm after the mTESE procedure, defined as the detection of at least one spermatozoon using an inverted microscope (× 40 lens, IX 71, Olympus, Japan). Secondary endpoints for cases with sperm retrieved and used in a subsequent ICSI cycle included the rates of fertilization (oocytes with two pronuclei as a function of the number of MII oocytes injected), blastocyst formation (the proportion of oocytes with two pronuclei that had formed a blastocyst by days 5/6), and ongoing pregnancy (fetal heartbeat at ≥ 12 weeks of gestation per oocyte retrieval).
Statistical analysis
Baseline characteristics were summarized as mean and standard deviations (SDs), median and interquartile range, and number with percentages, where appropriate. We performed all comparisons using the cluster bootstrap regression model. For the ICSI cycles, we analyzed the embryo development at the per woman and per oocyte level. We performed a backward comparison of the regression model to identify the confounders to be included. We found the model analyzing embryo development and clinical pregnancy should be adjusted for maternal age as covariate. The model comparing the presence or absence of sperm in testicular biopsies was adjusted for the paternal age as covariate after standardization. At the per woman level, fertilization and embryo development were compared using Poisson regression analysis, summarizing the effect size as relative risk (RR) with 95% confidence intervals (CIs). At the oocyte level, fertilization and embryo development were compared using logistic regression, summarizing the effect size as odds ratio (OR) with 95% confidence intervals (CIs). We used the R Statistical Package [19] for all analyses.
