Can sperm quality influence embryo development and its ploidy? Analysis of 811 blastocysts obtained from different sperm sources.

The 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.

How 1 h of abstinence improves sperm quality and increases embryo euploidy rate after PGT-A: a study on 106 sibling biopsied blastocysts.

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.

The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time.

OBJECTIVE: the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. MATERIALS AND METHODS: Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. RESULTS: With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. CONCLUSIONS: Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

Testicular histopathology, semen analysis and FSH, predictive value of sperm retrieval: supportive counseling in case of reoperation after testicular sperm extraction (TESE).

BACKGROUND: To provide indicators for the likelihood of sperm retrieval in patients undergoing testicular sperm extraction is a major issue in the management of male infertility by TESE. The aim of our study was to determine the impact of different parameters, including testicular histopathology, on sperm retrieval in case of reoperation in patients undergoing testicular sperm extraction. METHODS: We retrospectively analyzed 486 patients who underwent sperm extraction for intracytoplasmic sperm injection and testicular biopsy. Histology was classified into: normal spermatogenesis; hypospermatogenesis (reduction in the number of normal spermatogenetic cells); maturation arrest (absence of the later stages of spermatogenesis); and Sertoli cell only (absence of germ cells). Semen analysis and serum FSH, LH and testosterone were measured. RESULTS: Four hundred thirty patients had non obstructive azoospermia, 53 severe oligozoospermia and 3 necrozoospermia. There were 307 (63%) successful sperm retrieval. Higher testicular volume, lower levels of FSH, and better histological features were predictive for sperm retrieval. The same parameters and younger age were predictive factors for shorter time for sperm recovery. After multivariable analysis, younger age, better semen parameters, better histological features and lower values of FSH remained predictive for shorter time for sperm retrieval while better semen and histology remained predictive factors for successful sperm retrieval. The predictive capacity of a score obtained by summing the points assigned for selected predictors (1 point for Sertoli cell only, 0.33 points for azoospermia, 0.004 points for each FSH mIU/ml) gave an area under the ROC curve of 0.843. CONCLUSIONS: This model can help the practitioner with counseling infertile men by reliably predicting the chance of obtaining spermatozoa with testicular sperm extraction when a repeat attempt is planned.

Influence of human sperm origin, testicular or ejaculated, on embryo morphokinetic development.

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.

Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.

STUDY QUESTION: Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTCIPANTS/MATERIALS, SETTING, METHOD: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: No specific funding was obtained for this study. None of the authors have any competing interests to declare.

A novel stepwise micro-TESE approach in non obstructive azoospermia.

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.

Analysis of MYO-Inositol effect on spermatozoa motility, in hyper viscous ejaculates and in patients with grades II and III varicocele.

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.

Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on 1128 sibling human zygotes.

PURPOSE: The aim of the present randomized, comparative study was to evaluate the effect of reduced culture volumes on sibling human embryo development. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Traditional intracytoplasmic sperm injection provides equivalent outcomes compared with human zona pellucida-bound selected sperm injection.

The 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.

Natural in vitro fertilization cycles.

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.

Sperm vacuoles negatively affect outcomes in intracytoplasmic morphologically selected sperm injection in terms of pregnancy, implantation, and live-birth rates.

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.

Successful management of a triplet heterotopic caesarean scar pregnancy after in vitro fertilization-embryo transfer.

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-embryo transfer 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 MEASURE(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.

Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa.

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.