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SummaryThe aim of our study was to evaluate the correlation between sperm quality and ploidy status of the derived blastocysts. We performed a retrospective analysis on a restricted pool of patients enrolling only those who had no female factors. Male patients with genetic factors affecting spermatogenesis were also excluded. We chose a maternal age ≤38 years to decrease the female factor, therefore the male factor was the main component of sterility. We divided the patients in four groups based on semen quality and comparing fertilization, pregnancy and euploidy rates above all. In total, 201 intracytoplasmic sperm injection (ICSI) cycles were enrolled in the study. Cycles were divided into four groups, according to semen source: normal semen, oligoasthenoteratozoospermia (OAT), cryptospermia or non-obstructive azoospermia (NOA). An extremely statistically lower fertilization rate was found in NOA patients. Unexpectedly, no differences were detected in blastocyst formation, euploidy, aneuploidy and mosaicism rates among the four groups. Interestingly, we also found a higher abortion rate comparing NOA to normal semen with an odds ratio of 4.67. In our study no statistically significant differences among the analyzed groups were found, showing little or no effect at all using spermatozoa from different semen sources or quality. This may be linked to the oocyte competence of fixing sperm DNA damage and it could be hypothesized that only sperm with a good rate of DNA integrity are able to fertilize the oocyte, explaining why poor quality semen is reflected in a low fertilization rate without effect on ploidy.

PURPOSE: The aim of our study was to evaluate the influence of different ejaculatory abstinence time frames (several days versus 1 h) on semen parameters, blastocysts ploidy rate, and clinical results in assisted reproduction cycles on sibling oocytes. METHODS: This is a prospective study including 22 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between November 2015 and December 2018. Male partners with oligoastenoteratozoospermia produced two semen samples on the day of oocyte retrieval: the first one after several days of abstinence and the second, 1 h after the first one. Oocytes from each patient were divided into two groups: those in group 1 were injected with spermatozoa from the first ejaculate (N = 121) and oocytes in group 2 with spermatozoa from the second one (N = 144). Outcomes of aniline blue test, fertilization, blastocyst formation, ploidy rates, and clinical results were compared between the two groups. RESULTS: Semen volume resulted lower in the second sperm retrieval. Sperm concentration, motility, and morphology were similar in the two groups. A total of 106 blasotcysts were biospied. Higher blastocyst euploidy rates resulted in group 2 (43.6%) than in group 1 (27.5%). A higher percentage of mature chromatine was observed in group 2. CONCLUSION: Using spermatozoa from samples with a shorter abstinence could be a simple method to select higher quality spermatozoa, reducing aneuploidy rate in blastocysts. Prospective randomized controlled trials should be performed to confirm the potential advantage of using semen samples with short abstinence period to improve the outcome of assisted reproduction cycles.

Study question Can MACS increase euploid blastocyst rate in Pre-implantation Genetic Testing (PGT) cycles for AMA-APA (Advanced Maternal-Paternal Age) in patients with high sperm DNA fragmentation (SDF)? Summary answer A slight increase in euploid blastocyst rate was found using MACS in infertile patients with high SDF undergoing PGT cycles compared to the control group. What is known already Many authors have shown a close correlation between the presence of apoptotic markers on spermatozoa and the failure of assisted reproduction treatments. In normal physiological conditions, apoptotic spermatozoa with phosphatidylserine (PS) residues externalized on the plasma membrane, are eliminated along female genital tract, preventing oocyte fertilization. MACS eliminates apoptotic sperm whit PS residues using superparamagnetic microbeads conjugated with annexin V. This technique reduces the proportion of sperm with high rates of SDF and can be used to maximize ART procedures results. MACS application improves sperm quality, fertilization, cleavage and pregnancy rates reducing miscarriage rate. Study design, size, duration From June to November 2020, 10 couples in which MACS was applied to select non-apoptotic spermatozoa, were randomly enrolled in our study (MACS group) and 8 couples without MACS were considered as controls (No-MACS Group). All couples in both groups underwent a PGT cycle and had high sperm DNA Fragmentation (> 20%). A higher rate of euploid and diploid-euploid mosaic blastocysts were obtained in the MACS group compared to the control group. Participants/materials, setting, methods Patients with severe oligoastenoteratozoospermia were excluded. MACS protocol was performed as follows: semen sample was analyzed (WHO 2010) and washed with buffered medium; pellet was removed and a swim-up was performed. Retrieved spermatozoa were washed with a binding buffer (Miltenyi Biotec), centrifuged (400 g x 4 minutes) and supernatant discarded. Pellet was covered with Annexin-V and re-suspended. After 15 minutes incubation at room temperature, the sample was eluted through the column and collected for ICSI. Main results and the role of chance In MACS group, female and male mean age ± SD were 41.6 ± 2.1 and 43.5 ± 7.3, respectively. Female and male mean age ± SD were 41.7 ± 2.8 and 44.6 ± 8.1 in the No-MACS group, respectively. In MACS and No-MACS groups, injected oocytes were 44 and 35, fertilized oocytes were 32 (72.3%) and 27 (77.1%) (NS), blastocyst formation rates were 71.8% (23/32) and 48.1% (13/27) (NS), respectively. In No-MACS group, only 1 euploid and 1 diploid-euploid mosaic blastocysts were obtained (1/13 = 8%) (NS). In MACS group, 4 euploid blastocysts were formed (4/23 = 17.4%) whereas mosaic diploid-euploid blastocysts were 3/23 (13.0%) (NS). Aneuploid blastocysts were 16/23 (69.6%) in MACS group and 11/13 (84.6%) in No-MACS group (NS). Limitations, reasons for caution AMA and APA of couples enrolled should be considered as a limit of the study. A larger number of patients and biopsied blastocysts are needed to analyze clinical results and perform a robust statistical analysis establishing if MACS is useful to improve transferable blastocyst rate in patients with high SDF. Wider implications of the findings: MACS is useful to select non apoptotic sperms; although fertilization, cleavage and blastocyst rates are not improved, aneuploid blastocysts rate slightly decreases using MACS. It I possible that, selecting spermatozoa free from PS residues, MACS allows to choose spermatozoa with a better DNA packaging, thus affecting the embryo ploidy. Trial registration number non applicable

Study question Could advanced paternal age influences the embryos aneuploidy rate in eggs donation cycles with poor sperm quality? Summary answer In case of severe male factors increased paternal age can affect embryos aneuploidy rate in egg donation cycles. What is known already While the impact of advanced maternal age on reproductive is well understood, the effect of paternal age on reproductive function is controversial. Many studies have shown that Advanced Paternal Age (APA) could impact on male fertility potential affecting testicular function and sperm quality. Moreover, APA also has been associated with increased epigenetics changes and DNA mutations. Increased paternal age could be associated with different types of disorders such as autism, schizopherenia and bipolar disorders. Egg donation cycles, controlling female variables, represent the ideal model for the study of the impact of paternal age on reproductive outcomes. Study design, size, duration We retrospectively analyzed 43 egg donation cycles (October 2014-January 2020) with ≥ 50% survival rate of vitrified/warmed oocyte. Only cycles with poor sperm quality were considered. Cycles were divided in two GROUPS: group–1 included male paternal age ≤ 45 while group–2 included male paternal age >45. Data, shown as avarage±SD, were analyzed with Chi square or Student-t test. Participants/materials, setting, methods Group–1 included 20 cycles and 219 oocytes, male age was 40,89 ±6.12; Group–2 included 17 cycles and 173 oocytes, male age was 51±6.06. Respectively, in Group 1 and in Group 2, donor age were 22.4±2.65 and 24.8±3.88 (NS). All oocytes were injected with abnormal sperm samples according to WHO 2010. Embryos were cultured in time-lapse system until blastocyst stage. Trophectoderm biopsy and PGT-A analysis were performed according to standardized laboratory protocols. Main results and the role of chance Oocytes survival rates in Group1 and 2 were 86% (188/219) and 90.7% (157/173) (NS), respectively. Fertilization rates in Group1 and –2 were 71.42 (135/189) and 73.45% (119/162) (NS), respectively. The total number of obtained embryos (transferred + frozen) were 81 and 801 in Group–1 and –2, respectively. The rates of obtained embryos per reiceved occytes were 37% (81/219) and 46.24% (80/173) in Group–1 and –2 (p Limitations, reasons for caution Each donor was stimulated with different protocols according to her history and hormones levels. Nothing is known about which type of sperm parameters (semen amount, morphology or motility) have a major impact when focusing on the embryos genetic outcome. Wider implications of the findings: To better known the effect of APA, it could be necessary identify embryos chromosomal abnormalities and the correlation with specific sperm parameters. Further studies should be done to confirm the APA effect in patients with severe male factors and define a cut-off male age where PGT-A should be recommended. Trial registration number Not applicable

The goal of this study is to evaluate MYOInositol effects on spermatozoa motility, in patients’ ejaculates with severe varicocele or hyper viscosity. The study included normal viscosity ejaculate from 30 patients affected by varicocele and hyper viscosity ejaculate from 33 patients without any testicular pathologies. All selected samples showed sperm concentration > 2 million/ml and progressive motility < 32%. In both groups, the pellet obtained after centrifugation in buffered medium, was divided in two aliquots, both incubated for 15 minutes at 37°C: one with MYO-Inositol and the other one, as control, only in phosphate buffered saline (PBS). Afterwards, the sperm progressive motility was assessed using Computer Assisted Sperm Analysis (CASA system). Incubation with MYO-Inositol improved sperm progressive motility in high viscosity samples compared to control group (38.9% ± 3.0 vs 24.35% ± 2.41, respectively; p ≤ 0.0001). Conversely, no statistically significant difference was observed in total sperm progressive motility in varicocele samples compared with control group (22.7% ± 2.07 vs 26.7% ± 3.31, respectively; p = 0.085). The MYO-Inositol positive effect on spermatozoa motility may depend on the type of sperm damage: heavy structural and biochemical defects which typically affects patients with varicocele are not restored by Inositol. On the contrary, MYOInositol is able to improve sperm motility in semen samples with high viscosity, since those samples show no substantial structural sperm defects.

The aim of the present randomized, comparative study was to evaluate the effect of reduced culture volumes on sibling human embryo development.Firstly, sibling injected oocytes obtained from 88 out of 165 consenting couples undergoing infertility treatment were cultured either in large (35 μl) or in small drops (15 μl) of culture medium. Secondly, sibling injected oocytes from 77 couples were cultured either in large (35 μl) or in mini drops (7 μl). Embryo quality on day-2 and day-3 and blastocyst formation rate on day-5 were evaluated.No statistically significant difference in terms of embryo quality was detected comparing embryos cultured either in large (35 μl) or small (15 μl) drops until blastocyst stage. Similarly, no difference appeared between large (35 μl) or mini (7 μl) drops until day-3, however a significantly higher blastocyst formation rate was observed in mini (7 μl) drops on day-5.Reduced culture volume seems not to influence early embryo development but a reduction of medium appears to positively affect blastocyst development. This supports the hypothesis that the pre-implantation embryo produces autocrine factors which exert a positive effect on embryo development when culture is performed in a reduced volume.

SummaryThe capability of human zona pellucida (ZP) to bind selectively to normal functional sperm with normal chromatin has been reported widely in the literature. The aim of this study was to evaluate whether ZP–binding sperm selection may represent a method to retrieve superior spermatozoa for intracytoplasmic sperm injection (ICSI). Patients were divided into two groups: a ZP-ICSI and a conventional ICSI group. In the ZP-ICSI group, spermatozoa for injection were selected after ZP–sperm incubation and spermatozoa that were tightly bound to the ZP were used for ICSI (ZP-ICSI). Clinical outcomes of ZP-ICSI were compared with the outcomes of traditional scientist-selected sperm injection (conventional ICSI). Results did not show any significant difference in fertilization, pregnancy, implantation and take-home-baby rates between conventional ICSI and ZP-ICSI. However, when data relative to patients who received ZP-ICSI were analyzed, an interesting result was observed: higher sperm concentration and morphology correlated with higher ZP–sperm binding. Additionally, patients with higher ZP–sperm binding seem to have improved pregnancy and take-home-baby rates. In conclusion, this study shows that ZP-ICSI is not a superior method compared with conventional ICSI. However, clinical ICSI outcomes were apparently improved in the presence of good ZP–sperm binding. We therefore speculate that sperm competence to ICSI could be reduced when the sperm's ability to bind the ZP is impaired.

Background The purpose of the study was to investigate whether micro-TESE can improve sperm retrieval rate (SRR) compared to conventional single TESE biopsy on the same testicle or to contralateral multiple TESE, by employing a novel stepwise micro-TESE approach in a population of poor prognosis patients with non-obstructive azoospermia (NOA). Methods Sixty-four poor prognosis NOA men undergoing surgical testicular sperm retrieval for ICSI, from March 2007 to April 2013, were included in this study. Patients inclusion criteria were a) previous unsuccessful TESE, b) unfavorable histology (SCOS, MA, sclerahyalinosis), c) Klinefelter syndrome. We employed a stepwise micro-TESE consisting three-steps: 1) single conventional TESE biopsy; 2) micro-TESE on the same testis; 3) contralateral multiple TESE. Results SRR was 28.1 % (18/64). Sperm was obtained in both the initial single conventional TESE and in the following micro-TESE. The positive or negative sperm retrieval was further confirmed by a contralateral multiple TESE, when performed. No significant pre-operative predictors of sperm retrieval, including patients’ age, previous negative TESE or serological markers (LH, FSH, inhibin B), were observed at univariate or multivariate analysis. Micro-TESE (step 2) did not improve sperm retrieval as compared to single TESE biopsy on the same testicle (step 1) or multiple contralateral TESE (step 3). Conclusions Stepwise micro-TESE could represent an optimal approach for sperm retrieval in NOA men. In our view, it should be offered to NOA patients in order to gradually increase surgical invasiveness, when necessary. Stepwise micro-TESE might also reduce the costs, time and efforts involved in surgery.

In this retrospective observational study (October 2014 - July 2016), the impact of sperm origin on embryo morphokinetics and on clinical outcomes after intracytoplasmic sperm injection was evaluated. The developmental kinetics of embryos obtained either with testicular sperm (40 cycles; testicular sperm group) or with thawed donor sperm (26 cycles; donor sperm group) was analysed up to day-3 of culture with a time-lapse incubation system. In the testicular sperm group, all patients were affected by nonobstructive azoospermia. The timing of second polar body extrusion (IIPB), and the time to reach the 4-cells (t4) and 9-cells (t9) stages, differed significantly between the two groups: the IIPB extrusion and t4 were anticipated, whereas t9 was retarded in the testicular sperm group. We hypothesise that a different sperm maturation grade may influence the timing of embryo development: an early paternal effect of testicular sperm could be manifested as an anticipation in the IIPB extrusion and in the time for reaching the 4-cells stage. Conversely, a later paternal effect could be visible as a retardation in the timing at which the embryo reaches the 9-cells stage. Interestingly, clinical outcomes did not differ between the two groups except the implantation rate which was significantly increased in the donor sperm group.

Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics?Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones.Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes.This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed.Mean female age was 36.8 ± 4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of the ICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing.Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showed top quality ICM (P 0.001), and 17.1% of euploid and 28.5% of aneuploid blastocysts showed poor quality ICM (P 0.001). Top quality TE was present in 46.5% of euploid and 31.1% of aneuploid blastocysts (P 0.001), while 26.6% of euploid and 38.1% of aneuploid blastocysts showed poor quality TE (P 0.001). Regarding expansion grade, 81.1% of euploid and 72.4% of aneuploid blastocysts were fully expanded (Grade 5-6; P 0.001). The timing of cleavage from the three- to four-cell stage, of reaching four-cell stage, of starting blastulation, reaching full blastocyst stage, blastocyst expansion and hatching were 2.6 (95% confidence interval (CI): 1.7-3.5), 40.0 (95% CI: 39.3-40.6), 103.4 (95% CI: 102.2-104.6), 110.2 (95% CI: 108.8-111.5), 118.7 (95% CI: 117.0-120.5) and 133.2 (95% CI: 131.2-135.2) hours in euploid blastocysts, and 4.2 (95% CI: 3.6-4.8), 41.1 (95% CI: 40.6-41.6), 105.0 (95% CI: 104.0-106.0), 112.8 (95% CI: 111.7-113.9), 122.1 (95% CI: 120.7-123.4) and 137.4 (95% CI: 135.7-139.1) hours in aneuploid blastocysts (P 0.05 for early and P 0.0001 for later stages of development), respectively. No statistically significant differences were found between euploid and aneuploid blastocysts for the remaining morphokinetic parameters.A total of 407 embryo transfers were performed (155 fresh, 252 frozen-thawed blastocysts). Higher clinical pregnancy, implantation and live birth rates were obtained in frozen-thawed compared to fresh embryo transfers (P = 0.0104, 0.0091 and 0.0148, respectively). The miscarriage rate was 16.1% and 19.6% in cryopreserved and fresh embryo transfer, respectively. The mean female age was lower in the euploid compared to aneuploid groups (35.0 ± 3.78 versus 36.7 ± 4.13 years, respectively), We found an increasing probability for aneuploidy with female age of 10% per year (odds ratio (OR) = 1.1, 95% CI: 1.1-1.2, P 0.001).The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performed morphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation.Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred.No specific funding was obtained for this study. None of the authors have any competing interests to declare.

Background Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication. Methods The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation. Results The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. Conclusions These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.

Objective To retrospectively evaluate whether sperm vacuoles influence clinical results, with a particular focus on live-birth rates, in 101 intracytoplasmic morphologically selected sperm injection (IMSI) cycles. Design Retrospective, observational study. Setting Medical center. Patient(s) A total of 101 couples with at least two failed intracytoplasmic sperm injection (ICSI) attempts and impaired sperm morphology. Intervention(s) Patients divided into two groups according to sperm morphology and vacuolization pattern: group A comprising patients with good quality spermatozoa (type I and/or type II spermatozoa) (n = 63 patients); group B comprising patients with low quality spermatozoa (type III and/or IV spermatozoa) (n = 38 patients). Main Outcome Measure(s) Fertilization rate, embryo quality, pregnancy, implantation, and live-birth rates. Result(s) No statistically significant differences were observed between group A and B with regard to "early" assisted reproduction outcomes (fertilization rate and embryo quality). However, the "late" outcomes (pregnancy, implantation, and live-birth rates) were statistically significantly higher in group A. Conclusion(s) These results confirm a correlation between sperm vacuoles and a negative IMSI outcome, suggesting that sperm vacuoles are related to the late paternal effect.

Objective To report a case of a triplet heterotopic caesarean scar pregnancy (CSP) with two gestational sacs implanted in the caesarean scar after in vitro fertilization-embryo transfer. Design Case report. Settings Private reproductive medicine center and obstetric department of a university hospital. Patient(s) A 31-year-old woman with previous caesarean section affected by secondary infertility related to male azoospermia. Intervention(s) In vitro fertilization-embryotransfer followed by early ultrasound diagnosis of heterotopic CSP and selective embryo reduction performed by transvaginal ultrasound-guided potassium chloride and methotrexate injection in the ectopic gestational sacs. Main outcome mesure(s) Successful pregnancy outcome. Result(s) An ongoing intrauterine pregnancy with a live birth after successful management of ectopic gestational sacs. Conclusion(s) Triplet heterotopic CSP with two ectopic gestational sacs may occur after IVF-ET and, to our knowledge, this is the first time such a complication has been reported. Ultrasound is the main tool allowing early diagnosis of this condition, and the injection of potassium chloride and methotrexate is a safe and efficacious treatment method. The gynecologist managing early pregnancy should be aware of the possibility of CSP, and patients must be appropriately counseled about the different treatment options.

Although the first in vitro fertilization (IVF) baby was born after a natural IVF cycle, very soon this procedure was almost abandoned mainly because of the very high cancellation rates, and controlled pharmacological ovarian hyperstimulation became the standard treatment in IVF cycles of normoresponder patients. However, in poor-responder patients, where only very few follicles can be recruited and very few oocytes, if any, can be retrieved after controlled ovarian hyperstimulation, natural IVF cycles may offer a comparable number of follicles, reduced costs, and less discomfort for the patients. In this group of patients, natural IVF cycle is a cost-effective approach.

Does the health status of infants fathered by nonmosaic Klinefelter syndrome (KS) patients whose partners underwent ICSI with sperm obtained from testicular dissection reveal any genetic risk for the offspring?.KS patients undergoing testicular sperm extraction (TESE) are capable of conceiving healthy children.Paternity has been successfully achieved in nonmosaic KS patients (47,XXY karyotype) by ICSI using either ejaculated or testicular spermatozoa. A crucial concern is the potential transmission of genetic abnormalities to the offspring. Some studies reported that 47,XXY spermatogonia are capable of completing spermatogenesis leading to the production of mature spermatozoa with increased aneuploidies. Other authors showed that where focal spermatogenesis is present in nonmosaic KS males, it originates from euploid germ cells and, therefore, produces normal mature gametes. In support of this finding, at present, the great majority of children born from nonmosaic KS patients are chromosomally normal.From April 2004 to June 2010, 38 azoospermic patients with nonmosaic KS were examined for the presence of testicular spermatozoa. Spermatozoa were retrieved from 15 patients and 26 ICSI cycles were done (16 with cryopreserved sperm). There were 15 pregnancies leading to the birth of 16 babies who were karyotyped at amniocentesis and after birth.Participants were recruited from couples attending the European Hospital, Rome, and Clinica MARGen, Granada, for infertility treatment. Both the European Hospital and Clinica MARGen are private clinics. Testicular tissue was extracted with TESE or micro-TESE. After retrieval, fresh sperm was used for ICSI or it was cryopreserved for future use.Spermatozoa were retrieved from 15 patients (14 TESE and 1 micro-TESE) out of 38 (39.5%). A total of 26 ICSI cycles were performed: 10 with fresh and 16 with cryopreserved-thawed sperm. Mean ages (y) of patients with positive and negative sperm retrieval were, respectively, 34.8 ± 1.72 and 35.6 ± 4.08 (NS, nonsignificant). Comparing ICSI cycles performed with fresh sperm (n = 10) to those performed with frozen-thawed sperm (n = 16): Fertilization rates per injected oocyte were 53.0% (44 of 83) and 47.8% (32 of 67), respectively (NS). The cleavage rate per injected oocyte was 90.6% (29 of 32) versus 68.2% (30 of 44); P = 0.026. Clinical outcomes were not significantly different between the fresh and the frozen-thawed sperm group: clinical pregnancy rates were 7 of 10 (70.0%) and 8 of 16 (50.0%); implanted embryos (per transferred embryo) were 8 of 23 (34.8%) and 8 of 29 (27.6%); delivery rates were 6 of 10 (60.0%) and 5 of 16 (31.3%). Sixteen babies were born, all of them are healthy with a normal karyotype, eight from the fresh sperm group and eight from the frozen-thawed sperm group.The small numbers available for study mean that only common problems can be excluded.This study provides further reassurance that KS men can father healthy children and that pre-implantation genetic diagnosis on embryos conceived with their sperm is not strongly indicated. However, until conclusive information is available, such couples should be offered extensive genetic counseling.No external funding was obtained for the present study. None of the authors has any conflict of interest to declare.Not applicable.