Key words
testicular sperm extraction - azoospermia - prognostic factors - male infertility
Schlüsselwörter
testikuläre Spermienextraktion - Azoospermie - prognostische Faktoren - männliche
Infertilität
Introduction
Azoospermia is defined as a total absence of spermatozoa in ejaculated semen. The
condition affects 1% of the male population and 10 – 15% of patients undergoing evaluation
for infertility [1]. Azoospermia is divided into non-obstructive azoospermia (no spermatozoa production)
and obstructive azoospermia (the absence or obstruction of ejaculatory ducts) [2]. Since the development of testicular sperm extraction (TESE), men with azoospermia
have a chance to father their own children by combining the procedure with intracytoplasmic
sperm injection (ICSI) [3]. Therefore, the aim of this study was to identify predictive clinical and biological
factors that contribute to the success rate of ICSI-TESE treatment and help to better
estimate individual chances of achieving pregnancy in couples with azoospermic men.
In subfertile men, chromosomal aberrations, such as Klinefelter Syndrome, and genetic
problems, such as cystic fibrosis transmembrane conductance regulator (CFTR) mutations
and azoospermia factor (AZF) deletions, are more prevalent compared with fertile men
[4], [5], [6], [7], [8].
The most common pathological finding in cases of non-obstructive azoospermia is tubular
atrophy, which leads to the impairment or arrest of spermatogenesis. The degree of
atrophy and impairment of spermatogenesis can be categorized using the scale proposed
by Sigg, which ranges from slight atrophy with hypospermatogenesis (SIGG 1) to complete
fibrosis and lack of spermatogenesis (SIGG 5) [9]. Due to the presence of variegated atrophy, the degree of atrophy can differ depending
on the cross section that is assessed [10].
In vitro fertilization (IVF) is a well-established treatment for a lot of cases of
infertility. However, in cases with extremely low sperm counts, impaired motility,
and poor morphology, fertilization can only be achieved by ICSI, which has been used
for more than 20 years [11]. In men with azoospermia, spermatozoa can be retrieved from testicular tissue samples;
however, reported retrieval rates differ, ranging from 41 to 86.6% [12], [13]. This may be due to differences in patient selection resulting in an overestimation
of retrieval rates [3], [14]. Furthermore, reported ICSI outcomes vary widely, mainly due to selection bias [3], [15]. There is little data available on pregnancy rates after TESE/ICSI [17].
Embryos are chosen for uterine transfer according to the timing and rate of cell division
and morphology. Factors considered in the grading of embryo quality (day 2 – 3) include
cell number, degree of fragmentation, cell symmetry, and the presence/absence of cytoplasmic
pitting [19]. In addition, blastocyst classification (on day 4 – 5) includes stage of development,
inner cell mass, and trophectoderm cells [19]. Implantation and live-birth rates have been reported to be associated with the
morphology of embryos and blastocysts [19], [20], [21].
Clinical parameters such as weight and smoking status are associated with an impact
on the outcomes following assisted reproductive techniques. For example, obesity and
smoking negatively influence male fertility by impairing semen parameters [22], [23], [24].
Patients, Material and Methods
Patients, Material and Methods
Study aim and design
Little is known about the clinical factors that affect the success of ICSI among couples
undergoing treatment following sperm-positive testicular biopsy. This retrospective
study aimed to assess the significance of various clinical and biological parameters
relevant to the outcomes of TESE/ICSI treatment. The primary outcome parameter was
the pregnancy rate; the secondary outcomes were the effects of age, BMI, smoking,
histological results, fertilization rate and rate of embryo transfers on the pregnancy
rate.
Participants
We recruited and included all couples who gave their consent after attending the Department
of Gynecological Endocrinology and Fertility Disorders, University Womenʼs Hospital
Heidelberg, Germany, for TESE/ICSI treatment from January 2008 through October 2015.
Exclusion criterion was the complete absence of sperm after testicular biopsy, which
prevented TESE/ICSI treatment and could therefore not lead to pregnancy.
Data collection
We collected data on age, body mass index (BMI), nicotine consumption of both partners
at the time of first consultation, testicular pathology after TESE procedure, fertilized
oocytes, transferred embryos, embryo quality, and pregnancy rates by reviewing the
medical records. Pregnancy was defined as a positive pregnancy test two weeks after
oocyte pickup and assessed by serum hCG 14 days after oocyte pickup (HCG > 10 mIU/ml = positive).
Abortion was included in the evaluation if it occurred within the first 6 to 9 weeks
of gestation. Pregnancies were followed up by ultrasound imaging during the period
starting two weeks after the positive pregnancy test, and patients were then referred
to a specialist for further prenatal care after detection of a positive heartbeat
(ongoing pregnancy rate). Further follow-up was not covered by this study. The cumulative
pregnancy rate was calculated based on the first 4 fresh cycles with embryo transfer
that were performed per couple
(including all commenced treatment cycles) and all positive pregnancy tests were
included in the evaluation. Only two couples underwent 5 or more fresh cycles.
According to the patient management protocol, azoospermia was diagnosed based on evidence
from two spermiograms.
Testicular sperm extraction
Couples were advised to seek genetic counseling prior to TESE, during which they were
informed about the option of TESE/ICSI treatment.
The procedure was performed in the Urological University Hospital, Heidelberg under
general anesthesia. Testicular tissue was removed using the “no-touch technique” to
avoid mechanical damage to tissue and artifacts for the histological examination [25]. Five biopsies were taken from each testis, and one sample from each side was used
for histological examination at the Pathology Institute of the University Hospital,
Heidelberg. All other testicular biopsies were taken to the IVF Laboratory of the
University Womenʼs Hospital, Heidelberg. The presence of sperm in tissue was evaluated
by microscopy (Nikon Ti-S inverse microscope at 200 – 400-fold magnification) in a
small piece from each sample, which was enzymatically digested by incubation with
600 IU collagenase (Collagenase Type IA, Sigma, C2674) at 37 °C for 1 – 3 hours.
Cryopreservation of testicular tissue
Testicular biopsies were cryopreserved using the Biological Freezing System Type BV-65
(Consarctic GmbH, Schöllkrippen, Germany). Before freezing, cryoprotective medium
(Sperm Freeze®, Ferti Pro, Berlin, Germany) was added to the samples. After stepwise freezing down
to − 196 °C, biopsies were stored in liquid nitrogen.
Before ICSI, a cryopreserved sample was thawed in a water bath at 37 °C for approx.
3 minutes. The testicular tissue was briefly washed in sperm preparation medium (SPM,
Origio, 107 000 60A), enzymatically dissociated with 600 IU collagenase (Collagenase
Type IA, Sigma, C2674) at 37 °C for 1 – 4 hours or overnight and washed twice in SPM.
Intracytoplasmic sperm injection
When sperms were identified in the testicular biopsy, the female partner underwent
stimulation for ICSI. Ovarian stimulation was performed as previously described [26]. The appropriate protocol and the cumulative dose of gonadotropins were determined
based on patientsʼ responses and the decisions made by the treating physicians. Women
were treated with one of the following:
-
the long-stimulation protocol, involving downregulation by administration of a GnRH
analogue (Synarela®, Pharmacia GmbH, Pfizer) followed by recombinant follicle-stimulating hormone (Gonal
F®, Merck Pharma GmbH, Darmstadt, Germany) or urinary hMG preparation (Menogon®, Ferring GmbH, Kiel, Germany); or
-
the short protocol, in which downregulation was induced with a GnRH analogue from
day 2 of the cycle; or
-
the antagonist protocol, starting with stimulation with gonadotropins on day 2 or
3, and administration of a GnRH antagonist from day 5 to 7 (Orgalutran®, MSD Merck, Sharp & Dohme Corp. or Cetrotide®, Merck Germany) [26].
As soon as at least three follicles reached a diameter of 17 – 18 mm, ovulation was
triggered (Ovitrelle®, Merck Pharma GmbH or Predalon®, MSD Sharp & Dohme GmbH). This was followed 36 – 38 hours later by transvaginal ultrasound-guided
follicular puncture under general anesthesia. ICSI was performed on mature oocytes
in Sydney IVF Gamete Buffer (K-SIGB-50; Cook Medical) with the sperm retrieved from
thawed testicular samples. The fertilization rate was evaluated after 16 – 18 hours,
as described previously [3]. Oocytes and embryos were cultured in vitro using sequential media with media changed
on day 3. The concentration of CO2 was set to adjust the pH of the culture medium within a range of 7.25 – 7.35. Culture
until day 3 was performed in Sydney IVF Cleavage Medium (K-SICM-20; Cook Medical)
covered with paraffin oil (10 100 060A; Origio). In the case of extended culture until
day 5, a change of media
was performed on day 3 of culture by replacing the spent medium with preequilibrated
Sydney IVF Blastocyst Medium (K-SIBM-20; Cook Medical). Cleavage stage embryos were
scored based on a modified grading scheme by Veek [27], which scored cell numbers, degree of fragmentation, and symmetry of cells. Grade
A included embryos with equally sized blastomeres and no fragmentation, grade B included
embryos with equally sized blastomeres and < 10% fragmentation, grade C included embryos
with unequally sized blastomeres and/or fragmentation of 10 – 50%, whereas grade D
included embryos with > 50% fragmentation. The grading scheme for cleavage stage embryos
was adjusted during the study period. The term “equally sized” was replaced with “stage-specific”.
The scoring of fragmentation was adjusted to grade A < 10%, grade B 10 – 25%, grade
C > 25 – 50% and grade D > 50%. On day 4 the initiation of compaction and morula formation
were
assessed. Blastocysts were scored based on the grading scheme by Gardner et al.
[28]. Briefly, the degree of blastocyst cavity expansion was assessed, with grade 1 achieving
< 50%, grade 2 achieving > 50% and grade 3 reaching up to 100% of the embryo volume.
Grade 4 included a cavity expansion of 100% embryo volume and thinning of the zona
pellucida of more than 50%. Grade 5 included hatching and grade 6 consisted of fully
hatched blastocysts. In addition, the quality of the inner cell mass (ICM) and trophectoderm
(TE) were assessed. The ICM of grade A consisted of many tightly packed cells. Grade
B consisted of several loosely grouped cells, and grade C consisted of very few cells.
The TE of grade A consisted of many cells forming a cohesive epithelium, grade B consisted
of a few cells forming a loose epithelium, and grade C consisted of very few large
cells.
High-quality embryos were defined as those with six to eight cells on day 3, or three
or four cells on day 2 respectively, classed as degree A and B. Poor-quality embryos
included those with fewer cells and/or classed as degree C or D. Classification of
blastocysts was carried out according to the developmental stage, formation of the
inner cell mass, and cell integrity of the trophectoderm [19]. Accordingly, they were defined as good quality embryos if they met the following
classifications ≥ 3AA, 1 – 2AA, 3 – 6AB and 3 – 6BA. Embryos classified as 1 – 6BC,
1 – 6CB, 1 – 6CC or 1 – 2BB were considered low quality embryos.
Statistical analysis
Continuous data are presented as mean ± standard deviation (SD) or minimum and maximum,
categorical data as absolute and relative frequencies (n, %). Associations were determined
using crosstabs and chi-squared tests, and the significance level was set at p < 0.05.
Statistical analyses were carried out using SAS/STAT® (SAS Institute, Inc., Cary, NC, USA).
Results
Demographics
We recruited 105 couples; of these, 15 were excluded due to no sperm in the biopsy.
Finally, 90 couples were enrolled and underwent a total of 168 ICSI cycles. A first
cycle was performed in all 90 couples, and oocytes were obtained from 87 (96.7%).
For 86 couples (95.6%), oocytes reached maturation (MII oocytes). Of these, MII-oocyte
fertilization with evidence of two pronuclei was achieved in 71 couples (82.6%). Embryo
transfer was carried out in 66 couples (73.3%). In the remaining 5 couples, embryos
had to be cryopreserved due to ovarian hyperstimulation syndrome or cervical stenosis.
The 66 transfers resulted in 21 pregnancies (pregnancy rate per transfer = 31.8%)
([Table 1]). A second cycle was performed in 42 couples. Oocytes were obtained from all 42,
and fertilization and embryo transfer were achieved in 35 couples (83.3%). Thirteen
pregnancies occurred after embryo transfer in these 35 couples (pregnancy rate per
transfer = 37.1%) ([Table 1]). A third cycle was performed in 19 couples. Oocytes were obtained from 18 couples
(94.7%); fertilization was achieved and embryo transfer was possible in 17 couples
(94.4%). Two pregnancies resulted among the 17 couples (pregnancy rate per transfer = 11.8%)
([Table 1]). A fourth cycle was performed in nine couples. Oocytes could be obtained in all
cases, fertilization was achieved in six couples (66.7%), and embryo transfer was
possible in five (55.6%). This resulted in one pregnancy (pregnancy rate per transfer = 20%).
These results are summarized in the flow chart in [Fig. 1]. Two couples underwent more than five cycles (16 cycles in total). Embryo transfer
was possible in 13 of the 16 cycles (81.3%), resulting in three pregnancies (cumulative
pregnancy rate per transfer = 18.8%). The mean numbers of oocytes, fertilization rate
and embryos per
couple of the first three cycles are detailed in [Table 1].
Table 1 Demographics: mean numbers of oocytes, fertilization rate, embryos per couple, abortion
and pregnancy rates are detailed in [Table 1].
|
Cycle 1 (90 couples)
|
Cycle 2 (42 couples)
|
Cycle 3 (19 couples)
|
|
ET = embryo transfer
|
|
Couples with follicles (n)
|
87/90 (96.7%)
|
42/42 (100.0%)
|
18/19 (94.7%)
|
|
Number of follicles per puncture (mean ± SD; min–max)
|
9.9 ± 8.0 (0 – 42)
|
10.8 ± 7.28 (2 – 32)
|
10.4 ± 7.4 (0 – 28)
|
|
Couples with MII cells
|
86/90 (95.6%)
|
42/42 (100.0%)
|
18/19 (94.7%)
|
|
Number of MII cells (mean ± SD; min–max)
|
7.5 ± 6.10 (0 – 40)
|
8.71 ± 5.78 (1 – 23)
|
7.8 ± 5.07 (2 – 21)
|
|
Couples with fertilization
|
71/86 (82.6%)
|
35/42 (83.3%)
|
17/18 (94.4%)
|
|
Fertilization rate (mean ± SD; min–max)
|
38.1 ± 29.8 (0 – 100)
|
32.5 ± 23.92 (0 – 88)
|
45.1 ± 30.0 (0 – 100)
|
|
Number of cells with 2PN (mean ± SD; min–max)
|
2.9 ± 3.7 (0 – 21)
|
3.0 ± 2.9 (0 – 11)
|
3.5 ± 3.1 (0 – 11)
|
|
Couples with embryo transfer
|
66/90 (73.3%)
|
35/42 (83.3%)
|
17/19 (89.5%)
|
|
Number of cells transferred (mean ± SD; min–max)
|
1.6 ± 0.5 (1 – 3)
|
1.6 ± 0.5 (1 – 2)
|
1.8 ± 0.4 (1 – 2)
|
|
Pregnancy rate/embryo transfer
|
21/66 (31.8%)
|
13/35 (37.1%)
|
2/17 (11.8%)
|
|
Pregnancy rate/couple
|
21/90 (23.3%)
|
13/42 (31%)
|
2/19 (10.5%)
|
|
Abortion/pregnancy (%)
|
4/21 (19.0%)
|
2/13 (15.4%)
|
0/2 (0%)
|
|
Ongoing pregnancy rate/ET (%)
|
17/66 (25.8%)
|
11/35 (31.4%)
|
2/17 (11.8%)
|
|
Ongoing pregnancy rate/couple
|
17/90 (18.9%)
|
11/42 (26.2%)
|
2/19 (10.5%)
|
Fig. 1 Flow chart diagram summarizing the results of the first four cycles of evaluated
couples.
The cumulative rate of a positive pregnancy test per embryo transfer for the first
4 fresh cycles in our study population was 56% (37/66); per couple we had a cumulative
rate of positive pregnancy tests of 41% (37/90) in the first 4 cycles. The latter
includes all couples who started the treatment, including the ones without embryo
transfer. For the ongoing pregnancy rates, we found a cumulative pregnancy rate for
the first 4 fresh cycles of 47% (31/66) per cycles with embryo transfer or 34.4% (31/90)
per couple, respectively.
For all included couples during all cycles, supernumerary embryos were cryopreserved
for later embryo transfers. Results of transfers after the cryopreservation of embryos
were not assessed.
Age, BMI and pregnancy rate
In the first cycle, pregnancy rates were higher when the male partner had a lower
BMI (p = 0.023), and for men who smoked (58.3%) compared with non-smokers (27.9%,
p = 0.05) ([Table 2]). It should be noted that among non-smokers, more patients suffered from higher-grade
SIGG testicular atrophy than smokers (Tables S1 and S2). Pregnancy rates showed no statistically significant relationship with BMI or smoking
status for the second or third cycles. The mean ages of both men and women were not
significantly related to treatment outcome ([Table 2]).
Table 2 Age and BMI. Pregnancy rate depending on age and BMI of both partners during the
first three cycles.
|
Pregnancy
|
Cycle 1
|
Cycle 2
|
Cycle 3
|
|
* Pregnancy yes X no, T-test. p = 0.023
|
|
BMI man (kg/m2) [range 19 – 34]
|
Yes
|
25.5 ± 2.8 (n = 19)
|
26.9 ± 4.7 (n = 11)
|
32.1 ± 7.4 (n = 2)
|
|
No
|
28.7 ± 5.9 (n = 35)*
|
27.6 ± 3.3 (n = 18)
|
26.7 ± 2.9 (n = 12)
|
|
Age of man (years) at TESE (mean ± SD) [range 28 – 73 yrs.]
|
Yes
|
37.1 ± 7.3 (n = 21)
|
38.3 ± 6.1 (n = 13)
|
36.2 ± 0.2 (n = 2)
|
|
No
|
38.8 ± 5.8 (n = 43)
|
38.8 ± 5.5 (n = 22)
|
38.7 ± 4.4 (n = 15)
|
|
BMI woman (kg/m2) [range 17 – 44]
|
Yes
|
24.7 ± 7.0 (n = 21)
|
23.7 ± 7.5 (n = 13)
|
25.5 ± 8.7 (n = 2)
|
|
No
|
24.3 ± 4.1 (n = 43)
|
24.3 ± 4.2 (n = 22)
|
25.2 ± 3.7 (n = 15)
|
|
Age of woman (years) at TESE (mean ± SD) [range 23 – 44 yrs.]
|
Yes
|
33.1 ± 4.4 (n = 21)
|
34.0 ± 4.6 (n = 13)
|
38.5 ± 2.2 (n = 2)
|
|
No
|
34.4 ± 4.9 (n = 45)
|
35.6 ± 3.7 (n = 22)
|
36.0 ± 3.7 (n = 15)
|
Pathological findings, smoking and pregnancy rate
Patients who had planned to undergo TESE/ICSI but in whom no sperm was detected were
not recorded in the IVF laboratory of the University Womenʼs Hospital Heidelberg until
2010. Therefore, only 15 patients with negative sperm specimens were recorded between
2010 and 2015. Of these, 26.7% were classified as SIGG grade 2, 20.0% as grade 3,
26. 7% as grade 4 and 26.7% as grade 5, which was higher than men who had sperm-positive
biopsies (Table S2 details SIGG grades of men with sperm-positive biopsies). In all cycles, the highest
rates of fertilization and pregnancy were achieved in men who did not exhibit pathological
findings ([Table 3]). Fertilization could not be achieved for 15 couples. The histological findings
of the men in these couples were as follows: No pathology was available for two (13.3%);
no atrophy was observed in five (33.3%); and two (13.3%), three (20.0%), two (13.3%),
and one (6. 7%) were classified as SIGG grade
1, 2, 3, and 4, respectively.
Table 3 Pathological findings and pregnancy. Results of histopathological analysis, fertilization
and pregnancy rates for the first three cycles.
|
Cycle 1
|
Cycle 2
|
Cycle 3
|
|
Fertilization (% ± SD, min–max)
|
Pregnancy rate*
|
Fertilization (% ± SD, min–max)
|
Pregnancy rate*
|
Fertilization (% ± SD, min–max)
|
Pregnancy rate*
|
|
* Pregnancy rate was calculated per couple with embryo transfer.
|
|
No pathological examination
|
43.9 ± 31.9 (0 – 100)
|
3/12 (25%)
|
45.4 ± 19.2 (25 – 75)
|
1/5 (20.0%)
|
33.0 ± 0
|
0/1 (0%)
|
|
No atrophy
|
43.1 ± 29.8 (0 – 100)
|
9/29 (31.0%)
|
34.94 ± 27.7 (0 – 88)
|
7/13 (53.9%)
|
58.0 ± 33.1 (0 – 100)
|
1/6 (16.7%)
|
|
SIGG 1
|
38.9 ± 34.6 (0 – 100)
|
5/9 (55.6%)
|
34.5 ± 22.0 (20 – 67)
|
1/4 (25.0%)
|
–
|
0/0 (0%)
|
|
SIGG 2
|
26.8 ± 26.0 (0 – 75)
|
2/8 (25.0%)
|
25.4 ± 17.4 (0 – 53)
|
3/7 (42.9%)
|
38.3 ± 36.5 (13 – 100)
|
0/4% (0%)
|
|
SIGG 3
|
23.0 ± 22.6 (0 – 56)
|
2/5 (40.0%)
|
20.6 ± 27.0 (0 – 64)
|
1/3 (33.3%)
|
35.7 ± 17.6 (17 – 52)
|
1/3 (33.3%)
|
|
SIGG 4
|
38.3 ± 30.6 (0 – 75)
|
0/3 (0%)
|
33.7 ± 30.6 (7 – 67)
|
0/3 (0%)
|
37.7 ± 23.4 (17 – 63)
|
0/3 (0%)
|
Embryo quality and pregnancy rate
The majority of embryos transferred were day 2 or 3 embryos. Table S3 lists the distribution of transferred embryos in terms of quality and time point
of transfer. Pregnancy rate following transfer was significantly affected by the number
and quality of the transferred embryos ([Table 4 a]). The highest pregnancy rate was observed after the transfer of two good quality
embryos in the first cycle (p = 0.003). An analysis of transfers after subdividing
into day 2/3 or 4/5 showed no significant differences in pregnancy rates between groups
([Table 4 b] and [c]).
Table 4 Pregnancy rate per transfer and embryo quality.
|
Cycle 1
|
Cycle 2
|
Cycle 3
|
|
a All embryos day 2/3 and day 5 cumulative
|
|
Embryo quality
|
n = 66
|
n = 35
|
n = 17
|
|
|
8/29 (27.6%)
|
6/17 (35.3%)
|
0/7 (0%)
|
|
|
10/18 (55.6%)
|
5/10 (50%)
|
2/7 (28.6%)
|
|
|
1/19 (15.8%)
|
2/8 (25%)
|
0/3 (0%)
|
|
p-value
|
0.003*
|
0.5384
|
0.289
|
|
b day 2/3 embryos
|
|
Embryo quality day 2/3
|
n = 57
|
n = 28
|
n = 1 715
|
|
|
6/26 (23,1%)
|
4/13 (30,8%)
|
0/6 (0%)
|
|
|
7/13 (53,9%)
|
4/8 (50%)
|
1/6 (16,7%)
|
|
|
3/18 (16,7%)
|
1/7 (14,3%)
|
0/3 (0%)
|
|
p-value
|
0.056
|
0.332
|
0.448
|
|
c day 5 embryos
|
|
Blastocyst quality day 4/5
|
n = 9
|
n = 7
|
n = 2
|
|
|
2/3 (66,7%)
|
2/4 (50%)
|
0/0
|
|
|
3/5 (60%)
|
1/2 (50%)
|
1/1 (100%)
|
|
|
0/1 (0%)
|
1/1 (100%)
|
0/0
|
|
p-value
|
0.487
|
0.646
|
–
|
Genetic reasons
Genetic consultations and examinations were carried out in 90 men. A CTFR mutation
was identified in eight men (8.9%). The couples with men with CFTR mutations underwent
between 1 and 10 cycles. Five of the eight couples achieved pregnancies. In six of
eight cases, there were no pathological findings, and in two cases only mild pathological
findings (SIGG 1, 2) were detected in the biopsies.
Discussion
This study evaluates the predictive clinical and biological factors that contribute
to the chance of pregnancy in couples with azoospermic males undergoing TESE/ICSI
treatment. Preexisting clinical factors prior to any TESE/ICSI treatment as well as
clinical parameters during that treatment were considered.
The present study reveals that the pregnancy rate after TESE depends significantly
on the quality of the embryo. The more qualitatively good embryos that are transferred,
the higher the chance of pregnancy. It has previously been demonstrated that the transfer
of high-quality embryos increases implantation and pregnancy rates after IVF [29], while the transfer of low-quality embryos results in higher miscarriage and lower
ongoing pregnancy rates [30]. Thus, embryo quality appears to be an important prognostic factor for the outcome
of ICSI after TESE as well. In the present study, this significant difference was
no longer observed when patients were divided into embryo transfer on day 2/3 and
day 4/5; this may be due to the limited number of patients. Additionally, the p-value
of the pregnancy rate in the first cycle almost reached significance for day 2/3 embryos
(0.056); and for blastocyst transfers, only a
single transfer with a poor-quality embryo was identified.
Our results indicate that the ongoing pregnancy rate decreases significantly by the
third cycle of TESE/ICSI. This clearly shows that the chance of pregnancy decreases
over time, possibly due to the fact that patients with a poorer prognosis often require
multiple cycles. Our cumulative rate for a positive pregnancy test was 56%, with an
ongoing pregnancy rate of 47% per embryo transfer, and 41%, or 34.4% respectively
for all couples treated after 4 fresh cycles. These results are in accordance with
other studies that found comparable cumulative pregnancy rates after IVF/ICSI treatment
[46] and suggest that TESE per se, when sperm are found within the biopsy, does not further
negatively affect patient outcomes.
Obesity and overweight status are global public health issues and a major clinical
concern [31]. Reduced fertility has been associated with overweight status and obesity in men
[32], with overweight men exhibiting lower sperm quality and reduced sperm counts compared
with normal-weight men [33], [34]. Additionally, the molecular structure, i.e., the miRNA profile in spermatozoa and
testicular somatic cells which affects sperm maturation and function, has been described
as altered in overweight patients [35]. Weight loss has been suggested to improve semen quality and fertility [36]. We observed a significant association between male BMI and pregnancy rate, which
is consistent with previous studies which showed that couples with lower-BMI men had
higher pregnancy rates [37]. This is also reflected in the guidelines that advise weight reduction in obese
men prior to ART [38]. Higher BMI may lead to increased temperature in obese menʼs testes [39], [40], and oxidative stress in the testes can lead to DNA fragmentation and mitochondrial
dysfunction, which negatively affects sperm quality and motility [41].
The women in our study had in median normal BMIs. Obesity is associated with lower
rates of live births after IVF and ICSI, along with impaired responses to ovarian
stimulation [2], [43]. Furthermore, increased BMI in women may have a negative effect on embryo quality
and, therefore, on the outcome of assisted-reproduction treatment [44].
Our study found that male age did not influence fertilization, embryo cleavage, or
pregnancy rate, which is consistent with previous research [45]. However, another study reported significant correlations between male age and pregnancy
and birth rates [47]. This is likely due to decreases in sperm count, motility, vitality, and morphology
that occur with increased age. In our study, female age had no significant impact
on the outcome of TESE/ICSI. This may be due to the homogeneous age distribution of
our population, since female age is one of the most important factors associated with
the success of ICSI [46].
Smoking is a potential risk factor for infertility, and has a negative effect on sperm
quality [48], [49], particularly sperm density, count, and morphology [50]. Nicotine use by women may adversely affect the outcome of IVF/ICSI treatment, due
to decreased endometrial thickness on the day of embryo transfer [51]; reduced rates of fertility, pregnancy, and live birth; and a higher risk of miscarriage
[52]. However, we did not find smoking to be associated with worse outcomes, probably
because the underlying pathological findings were milder among those who smoked.
In the present study, men were subdivided into groups according to their pathological
TESE findings. Although no pathological examinations had been documented for 14 men,
mature spermatozoa were found in all of these men. Of the remaining 76 patients, mature
spermatozoa were found in patients categorized as SIGG grades 1 – 4. Although finding
sperm in patients categorized as SIGG 4 is not expected, a fertilization rate of 38.3 ± 30.6%
was achieved in those couples where menʼs biopsies were diagnosed as SIGG 4. This
is consistent with a recently published study which showed that 14% of patients with
Sertoli-Cell-Only Syndrome (SIGG 4) and sperm-positive TESE were able to proceed to
ICSI [50]. In contrast, in 26.6% of patients categorized as SIGG 2, sperm could not be found.
It has been suggested that once spermatozoa are found, fertilization and pregnancy
rates do not differ significantly with regard to SIGG grade [53]. However, this was not the case in the present study. The presence of sperm tubules
with spermatozoa in the TESE biopsy is an important predictor of successful sperm
retrieval [54]. Classification as SIGG grade 5 was the only pathological finding associated with
no detectable sperm in our population. Therefore, our results indicate that the degree
of testicular atrophy other than total fibrosis (SIGG 5) is not prognostic for the
detection of mature sperm. Nevertheless, no pregnancies were achieved for patients
categorized as SIGG grade 4, which could be postulated as a negative predictive factor
for therapy success in those couples.
The highest pregnancy rates were observed in the present study when no atrophy or
pathology were documented, as is the case after vasectomy, or when only slight atrophy
(SIGG 1) was described. Regardless of the detection of mature sperm, a diagnosis of
SIGG grade 4 was associated with no pregnancy. To summarize, higher testicular atrophy
is associated with lower pregnancy success rates after TESE/ICSI.
Strengths and limitations
Our study found that smoking had no significant impact on histological findings and
there was no correlation between histology and outcome parameters such as pregnancy
rates and fertilization, unless very severe histological aberrations (≥ SIGG 4) were
present. These results are similar to those of another study of 86 men with non-obstructive
azoospermia pregnancies for all histological groups except those with maturation arrest
[55]. As could be expected, embryo quality as well as BMI of the male partner were found
to be significantly associated with pregnancy after treatment. The limitations of
this study are the retrospective nature of the study, that it was a single center
analysis, and the limited number of patients. Additionally, information about delivery
rates was limited [16] and was not evaluated here.
Our results can contribute to careful counseling offered to patients prior to TESE/ICSI
treatment. However, the total number of analyzed patients was small and even smaller
for each subgroup. This is also reflected in the results of the impact of BMI that
was only found to significantly affect the outcomes of the first fresh cycle and not
the following cycles.
Conclusion
In conclusion, embryo quality significantly influences the rate of successful pregnancies
in patients undergoing TESE/ICSI. The success rate is further affected by the BMI
of the male partner, with a lower BMI associated with a higher rate of conception
after TESE/ICSI treatment. The ongoing pregnancy rate decreased significantly after
the second attempt at treatment, while BMI and age of the female partner and nicotine
abuse by either partner did not affect the success rate of treatment. High-degree
testicular atrophy (SIGG grade 4) is a poor prognostic factor for the success of TESE/ICSI.
Considering these results, overweight men should be advised about weight optimization
prior to therapy. A realistic explanation for the success rates of TESE/ICSI should
take the histological results into consideration, even after sperm-positive biopsy.
Author Contributions
Each author contributed substantially to the drafting or critical revision of the
manuscript, and approved the final version. J. R., A. G., S. R. conceived and designed
the study. Data acquisition, data analysis and interpretation, and statistical analysis
were performed by T. B., E. C., J. H., J. R., S. R., T. S. and A. G. The sperm count
after testicular biopsy was carried out by J. D.
Data Availability
The data are available from the corresponding author on reasonable request.
Ethical Approval and Informed Consent
Ethical Approval and Informed Consent
The study was conducted in accordance with the principles of the Declaration of Helsinki.
The project was approved by the Ethics Committee of the Medical Faculty of Heidelberg
University (# S361-2008). All patients gave their informed consent and completed a
clinical questionnaire.
