Manuscript
Introduction
Since 1992, intracytoplasmic sperm injection (ICSI) has been used to overcome many causes of male factor infertility, including cryptozoospermia.1 According to the World Health Organization (WHO) cryptozoospermia is defined as the absence of spermatozoa from fresh preparations but observed in a centrifuged pellet.2 Due to low sperm count (<103 spermatozoa/mL) observed in the seminal fluid after centrifugation, cryptozoospermic patients require assisted reproductive technology (ART) to achieve a pregnancy.3 Furthermore, men with cryptozoospermia may suffer from virtual azoospermia.4 Because of this, many clinicians may consider the use of testicular sperm extraction (TESE) to achieve a better ART outcome. 5,6
Controversy exists over the use of ejaculate versus testicular sperm for ICSI in cryptozoospermic patients.5-9 Ejaculate sperm is thought to be more mature than testicular sperm.10 Nevertheless, there are inherent concerns with the use of ejaculate sperm; the repeated centrifugations needed to identify viable sperm may increase the production of reactive oxidative species affecting the quality of the sperm.11 Moreover, it has been proposed that sperm could suffer DNA damage due to oxidative stress after the release from Sertoli cells leading to low sperm quality and impaired clinical outcomes.12-14 If DNA damage detected in ejaculated spermatozoa begins after the sperm is release from Sertoli cells, it can be hypothesized that sperm recovered directly from the testis could be less affected by this pathological process when compared with ejaculated sperm.
On the other hand, TESE has shown to have debatable benefits over ejaculate sperm,5-9 and it carries risks of surgical complications and long-term adverse effects including hypoandrogenism.15,16 Additionally, previous studies have described a correlation between testicular extracted sperm and spermatic aneuploidy in patients with non-obstructive azoospermia.17 However, there are currently no peer reviewed publications associating higher aneuploidy rates with cryptozoospermia.
Given the lack of information regarding the possible causes for suboptimal outcomes patients with cryptozoospermia, and the possible relationship of embryonic aneuploidy in embryos derived form TESE, we sought to determine whether the embryonic euploidy rate differs in blastocysts derived from testicular versus ejaculated sperm in cryptozoospermic patients.
Materials and Methods
Study design and patient population
This retrospective, single center study included all cryptozoospermic patients who underwent in vitro fertilization (IVF) with ICSI and preimplantation genetic screening for aneuploidy (PGT-A) at RMA NY using next generation sequencing, from 2014 through December 2019. Cases of patients harboring chromosomal rearrangements, undergoing preimplantation genetic testing for monogenic defects (PGT-M) and/or using donor gametes were excluded from the analysis. Cohorts were segregated based on the source of sperm (ejaculated versus testicular). Demographic characteristics such as age, BMI (body mass index), ovarian reserve metrics were collected. Cycle characteristics and embryologic data, including number of mature oocytes (MII)fertilization rate, blastulation rate (total number of viable blastocysts over the total number of fertilized oocytes), embryo quality and ploidy rates (number of euploid/aneuploid/indeterminate blastocysts over the number of biopsied blastocysts) were compared between cohorts.
Stimulation protocol
Patients underwent controlled ovarian hyperstimulation (COH) for IVF as previously described.18 Briefly, the COH protocol was selected at the discretion of the reproductive endocrinologist and involved the administration of follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) with a gonadotropin-releasing hormone (GnRH) agonist downregulation protocol with leuprolide acetate (Lupron, AbbVie Inc., North Chicago, IL), a GnRH antagonist protocol (Ganirelix Acetate, Organon USA Inc., Roseland, NJ or Cetrotide, EMD Serono, Rockland, MA), or a microflare protocol with leuprolide acetate (Lupron, AbbVie Inc., North Chicago, IL). These protocols have been described previously.18 Follicular development was monitored using transvaginal ultrasonography. When at least two follicles reached 18 mm in diameter, final oocyte maturation was induced with either hCG (5000–10,000 IU, Novarel, Ferring Pharmaceuticals, Parsippany, NJ, USA), recombinant human chorionic gonadotropin (250–500 μg, Ovidrel, EMD Serono, Rockland, MA) or, in high responders at risk of ovarian hyperstimulation syndrome undergoing a GnRH antagonist protocol, a dual trigger with 2 mg of leuprolide acetate and 1000 IU of Hcg or leuprolide acetate alone. Thereafter, patients underwent vaginal oocyte retrieval under transvaginal ultrasound guidance 36h after oocyte maturation was triggered.
Methodology of the TESE Procedure
Patients with cryptozoospermia underwent testicular sperm retrieval as previously described.20 Briefly, after stabilization of the testicle, a small incision in the testicle's midportion was performed, cutting through the scrotal skin, tunica vaginalis, and albuginea. A substantial piece of the extruding testicular tissue was cut with small scissors, washed with medium to remove blood traces, and placed in a Petri dish. Testicular tissue was vigorously fragmented and minced using two glass slides and immediately examined under the inverted microscope for the presence of spermatozoa in a wet preparation. Once spermatozoa were found, the surgical procedure was terminated. If spermatozoa were not observed, additional biopsies were taken from different areas of the same testicle and also from the contralateral one.
Laboratory procedures
Embryo Culture
All metaphase II (MII) oocytes underwent intracytoplasmic sperm injection (ICSI). Embryos were cultured to the blastocyst stage as previously described (Hernandez-Nieto, et al. 2019). On day 3 of embryo development, all embryos underwent laser-assisted zona hatching by creating a 25–30 µm opening in the zona pellucida with a 200– 300 ms pulse using ZILOS-tk Laser (Hamilton Thorne Biosciences, MA, USA) to facilitate posterior trophectoderm herniation. Blastocyst trophectoderm biopsies were performed on day 5-7 of development, contingent upon morphologic eligibility (Modified Gardner Scoring system).19 Biopsy was performed as described previously.18 The biopsy samples were placed in hypotonic wash buffer and submitted for analysis. Embryos were vitrified after the biopsies. Five to seven cells were analyzed by next generation sequencing (NGS) in order to determine chromosome, copy number and assigned to the following categories: euploid, aneuploid or inconclusive by the reference laboratory (during the study period mosaicism was not yet reported).
Outcome measures
The primary outcome was ploidy rate in blastocysts derived from testicular versus ejaculated sperm in cryptozoospermic patients, defined as the number of euploid and/or aneuploid blastocysts over the total number of biopsied blastocysts. Secondary outcome measures included fertilization rate, blastulation rate (total number of viable blastocysts over the total number of fertilized oocytes), and number of biopsied embryos.
Statistical analysis
Descriptive data was compared by Student’s t-test, Wilcoxon’ rank test and chi-square test when appropriate. The results were expressed as percentages, means and SDs with Clopper–Pearson binomial 95% CI. Adjusted odds ratios (OR) with 95% CI were calculated using multivariate logistic regression analyses to adjust for confounding variables. Logistic regression models were fitted with generalized estimating equations (GEE) to account for patients that underwent multiple cycles. Statistical analyses were performed using SAS version 9.4 (SAS institute Inc., Cary, NC, USA). All p-values were two-sided and were considered significant if less than 0.05.
Regulatory approval
This retrospective study was approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board, Inc.
Results
A total of 573 blastocysts derived from 87 IVF PGT-A cases were included in the study. 74 cases (n= 474 embryos) utilized ejaculated sperm and 13 cases (n= 99 embryos) utilized testicular sperm. No significant differences were found in demographic and cycle characteristics among cohorts. (Table 1)
|
Ejaculated sperm cycles |
Testicular sperm cycles |
|
|
N=74 |
|
N=13 |
|
|
|
Mean |
SD |
Mean |
SD |
|
| Male patient age (years) |
39.77 |
7.48 |
42.82 |
7.26 |
0.17 |
| Female patient age (years) |
36.13 |
4.36 |
36.98 |
5.29 |
0.53 |
| BMI (kg/m2) |
24.73 |
4.53 |
23.91 |
5.10 |
0.55 |
| Baseline FSH (IU/mL) |
6.01 |
4.03 |
4.80 |
2.46 |
0.36 |
| Antimullerian hormone (ng/ml) |
2.60 |
3.45 |
3.05 |
2.35 |
0.69 |
| Baseline Antral Follicle count |
10.81 |
6.05 |
12.70 |
3.92 |
0.34 |
| Surge E2 (pg/mL) |
2055.10 |
1036.86 |
2490.62 |
1215.28 |
0.17 |
| Mature MII oocytes |
10.12 |
6.50 |
12.46 |
7.15 |
0.24 |
| Fertilized oocytes |
6.41 |
4.57 |
7.62 |
6.28 |
0.40 |
| Total blastocysts / Cycle |
4.14 |
4.01 |
5.08 |
5.16 |
0.45 |
| Biopsied embryos /Cycle |
3.65 |
2.95 |
5.75 |
4.83 |
0.26 |
| Euploid embryos/ Cycle |
1.81 |
2.06 |
3.00 |
3.07 |
0.16 |
| Previous Oocyte Retrievals |
0.69 |
1.80 |
1.00 |
1.22 |
0.55 |
|
N |
% |
N |
% |
|
| Cancelled cycles / No embryos for Biopsy |
14/74 |
18.9 |
1/13 |
7.6 |
0.32 |
| Fertilization rate |
474/749 |
63.2 |
99/162 |
61.1 |
0.61 |
| Blastulation rate |
306/474 |
64.5 |
66/99 |
66.6 |
0.69 |
| Blastocyst biopsied/ Non biopsied Rate |
201/306 |
65.6 |
46/66 |
69.6 |
0.53 |
| Euploidy rate |
100/201 |
49.7 |
24/46 |
52.1 |
0.76 |
Table 1. Demographic cycle characteristics and laboratory outcomes of couples who suffer from cryptozoospermia and underwent an autologous IVF with PGT-A.
Note: Data presented as mean, percentages and standard deviation, unless stated otherwise. Abbreviations: BMI, body mass index; LH, luteinizing hormone; FSH, follicle‐stimulating hormone; MII, metaphase II. Significance established at p < .05.
No differences were found in fertilization rate (63.2%; 61.1%, p=0.32); blastulation rate (64.5%; 66.6%, p=0.69); and rate of embryo euploidy (49.7%; 52.1%, p=0.76) among cohorts. Finally, no differences were found in rate of cycle cancellation due to unavailable embryos for TE biopsy (18.9% vs 7.6%, p=0.32). (Table 1)
After adjusting for female and male patient’s age, BMI, AMH, and number of biopsied embryos, there were no association with surgical extracted sperm and lower odds of embryo euploidy (OR 0.69, CI95% 0.11-4.3, p=0.69).
Discussion
The results of this analysis suggest that there is no association with surgical extracted sperm and lower rates of embryonic euploidy. Whereas prior studies have investigated reproductive outcomes of fresh ejaculate or TESE and the transfer of unscreened embryos in patients with cryptozoospermia, this study is among the first to focus on the influence of the sperm source (from testicular versus ejaculated sperm) over the embryonic euploidy rate in cryptozoospermic patients.
When comparing ICSI outcomes using testicular versus ejaculate sperm in patients with cryptozoospermia in fresh embryo transfers, studies have yield divergent result.20,21 O’Connell et al. demonstrated ejaculated sperm to be more optimal than testicular sperms in cryptozoospermia patients and suggested that fertilization rates are related to sperm maturation.20 Conversely, Cui X et al. demonstrated that the use of testicular sperm achieved better embryonic quality and IVF outcomes than ejaculate sperm.21 Few studies have compared the fertility outcome of ejaculate with testicular sperm cells. Weissman et al reported a series of 4 couples with male factor infertility and multiple failed IVF/ICSI cycles with poor embryo quality and repeated implantation failure using motile ejaculatory sperm cells. The use of fresh testicular sperm cells resulted in better embryonic quality and pregnancies in all cases.22 Contrary to Weissman findings, our study found similar laboratory outcomes when utilizing both ejaculated or testicular sperm and ICSI.
Normal chromosomal composition is a primary driver of embryonic competence and reproductive success in patients undergoing ART. It is well established that the most important factor to achieve a euploid embryo is the oocyte’s age.23 However, the paternal genome also plays a crucial role.24 Because of this, there has been growing concerns regarding the possible chromosomal anomalies in offspring of men with severe male infertility.24 Particularly in embryos derived from testicular versus ejaculate sperm in spite of a young female partner.25 Even if the infertile male is chromosomally normal in his peripheral lymphocytes, a meiotic disruption may generate high rates of sperm chromosome abnormalities. Because of this, many researchers suggest evaluating the chromosomal complement of the spermatozoa in patients with severe male factor infertility and normal karyotypes.26 Multiple studies utilized fluorescence in situ hybridization (FISH) to assess the genetic composition of ejaculate sperm in comparison with surgically retrieved spermatozoa. One of the earliest reports studied chromosomes X, Y, 18 in the spermatozoa of 34 men with severe male factor infertility. The authors claimed that testicular spermatozoa presented higher rates of chromosomal aneuploidy in comparison with ejaculate sperm (19.6% vs 13%).27 In light of this, Cheung et al. compared sperm aneuploidy rates in ejaculated and testicular spermatozoa in the same individuals using FISH and NGS. After evaluating 9 chromosomes, the study reported that the total aneuploidy of surgically retrieved spermatozoa are comparable to that of ejaculated spermatozoa, corroborating that the use of testicular sperm is safe and does not increase aneuploidy rates.28 Our study findings are similar as we found no association with surgical extracted sperm and lower odds of embryo euploidy (OR 0.69, CI95% 0.11-4.3, p=0.69).
Our study distinguishes itself as it was performed at a single, high-volume academic center with a team of embryologists all uniformly trained, thereby reducing the inherent variability that may arise from multicenter studies. Patients with recognizable risk factors for poor embryonic development, such as parental chromosomal rearrangements, were excluded from the analysis, thus making our findings more generalizable. Aside from a large cohort, we use clinically validated PGT-A techniques to assess the rates of embryonic ploidy for all embryos analyzed, ensuring uniformity within the embryonic genetic results.
Notwithstanding our best efforts to avoid biases, some shortcomings and limitations exist in the analysis. The most notable limitation is its retrospective design, which increases the chance of selection bias. Furthermore, the number of patients that underwent a TESE is limited, However, the retrieval of testicular sperm mandates a surgical intervention and embedded risks. In light of the lack of data about the preferable source of sperm cells for ICSI in patients with cryptozoospermia, it would be unethical to design such a prospective research rather than to first use ejaculated sperm.
Conclusions
To our knowledge, this study is the first to evaluate the rate of embryonic euploidy in blastocysts derived from testicular versus ejaculated sperm in cryptozoospermic patients. Our analysis shows that there is no genomic advantage to surgical sperm retrieval in cryptozoospermic patients. Furthermore, we demonstrated that the use of testicular or ejaculate spermatozoa for ICSI can compensate for the reproductive disadvantage associated with the semen parameters of patients with cryptozoospermia. These data can be used to counsel patients about the chromosomal composition of embryos and ART outcomes derived when cryptozoospermia is encountered and to reassure them that the method of sperm collection prior to insemination via ICSI will not influence their IVF clinical success. Further randomized prospective studies should be performed in order to generate personalized and evidence-based recommendations for couples facing cryptozoospermia.
