The Fischer protocol for assisted reproductive technology treatment: Real-world data experience comparing elective single versus double embryo transfer with or without comprehensive chromosome screening.

High rates of multiple implantation after assisted reproductive technology (ART) treatment represent one of the major problems for both mothers and their fetuses. Given the availability of techniques intended to identify embryos with the highest chance for development to term, such as comprehensive chromosome screening (CCS) and blastocyst transfer, the decision on the number of embryos to transfer deserves careful consideration. This report presents real-life data from two clinics using the Fischer protocol for cycle programming in patients undergoing ART. Our data indicate that ovarian stimulation using the Fischer protocol provides consistent and optimal ART outcomes in centers following strict quality management standards. However, high multiple implantation rates were observed in fresh and frozen transfer cycles after transferring two embryos - even in patients aged over 39 years. The live birth rates after CCS were superior to those using untested embryos. These findings were held for the three age groups irrespective of the CCS culture day (D1 = PN stages, or D5 = blastocysts). Our results support a single embryo transfer policy, particularly in women under 34 years of age with favorable conditions during ART treatment, i.e., a high number of available fertilized oocytes.

SARS-CoV-2 pandemic and repercussions for male infertility patients: A proposal for the individualized provision of andrological services.

The prolonged lockdown of health facilities providing non-urgent gamete cryopreservation-as currently recommended by many reproductive medicine entities and regulatory authorities due to the SARS-CoV-2 pandemic will be detrimental for subgroups of male infertility patients. We believe the existing recommendations should be promptly modified and propose that the same permissive approach for sperm banking granted for men with cancer is expanded to other groups of vulnerable patients. These groups include infertility patients (eg, azoospermic and cryptozoospermic) undergoing medical or surgical treatment to improve sperm quantity and quality, as well as males of reproductive age affected by inflammatory and systemic auto-immune diseases who are about to start treatment with gonadotoxic drugs or who are under remission. In both scenarios, the "fertility window" may be transitory; postponing diagnostic semen analysis and sperm banking in these men could compromise the prospects of biological parenthood. Moreover, we provide recommendations on how to continue the provision of andrological services in a considered manner and a safe environment. Our opinion is timely and relevant given the fact that fertility services are currently rated as of low priority in most countries.

Intracytoplasmic morphologically selected sperm injection results in improved clinical outcomes in couples with previous ICSI failures or male factor infertility: a meta-analysis.

The objective of this study was to perform the first meta-analysis to compare conventional intracytoplasmic sperm injection (ICSI) outcomes and intracytoplasmic morphologically selected sperm injection (IMSI) outcomes in couples with previous ICSI failures (IF) or male factor infertility (MF). A systematic review was performed by searching Medline database to identify articles reporting on the comparison between ICSI and IMSI outcomes in couples with IF or MF. The main outcome measures were the implantation, pregnancy and miscarriage rates. Thirteen studies fulfilled our predetermined criteria. The overall results of meta-analysis for implantation (OR: 2.88; CI: 2.13-3.89), pregnancy (OR: 2.07; CI: 1.22-3.50) and miscarriage rates (OR: 0.31; CI: 0.14-0.67) were in favor of IMSI in couples with IF. Additionally, the overall result of meta-analysis for implantation (OR: 1.56; CI: 1.11-2.18) and pregnancy rate (OR: 1.61; CI: 1.17-2.23) were in favor of IMSI in couples with MF. IMSI increases the odds of implantation by 50% and pregnancy by 60% in couples with MF. In light of improved clinical outcomes, we recommend promoting the IMSI method in couples with MF. Moreover, IMSI results in a 3-fold increase in implantation rate, a 2-fold increase in pregnancy rate and a 70% decrease in miscarriage rate as compared to ICSI in couples with IF, however, as no randomized evidence exists, randomized studies are needed to confirm the IMSI benefits in couples with IF.

The importance of the cleavage stage morphology evaluation for blastocyst transfer in patients with good prognosis.

PURPOSE: To evaluate: (i) the influence of morphology at cleavage stage on blastocyst formation and implantation, and (ii) whether the transfer of low-quality embryos on day-three would be a better approach than the transfer at blastocyst stage. METHODS: This study included 8,444 embryos obtained from 1,125 patients undergoing ICSI cycles between January/2011 and September/2013. The influence of the quality of the embryo on days-two and -three on blastocyst formation and implantation success was evaluated. Moreover, the implantation potential of low-quality embryos, at cleavage stage, transferred on day-three was compared with the implantation potential of low-quality embryos, at cleavage stage, transferred on day-five. RESULTS: Low-quality embryos on day-two had an approximate 20 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-two had a nearly 40 % decrease in the implantation chance. Low-quality embryos on day-three had a 30 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-three had an almost 40 % decreased implantation chance. The implantation rate didn't differ when low-quality embryos on the cleavage stage were transferred on day-three or left in culture and transferred on day-five. CONCLUSIONS: The transfer of low-quality embryos on day-three is a better approach than transfer at the blastocyst stage. In addition, the embryo morphology evaluation at the cleavage stage is still needed for the selection of the embryo with the best implantation potential in extended embryo culture programmes.

Gender incidence of intracytoplasmic morphologically selected sperm injection-derived embryos: a prospective randomized study.

The aim of this prospective randomized study was to determine if the use of intracytoplasmic morphologically selected sperm injection (IMSI) is associated with gender incidence. Couples who underwent IVF-preimplantation genetic screening (PGS) cycles, as a result of advanced maternal age, were randomly allocated into two groups: intracytoplasmic sperm injection (ICSI; n=80) or intracytoplasmic morphologically selected sperm injection (IMSI; n=80). The incidences of genders were compared between ICSI- and IMSI-derived embryos. Considering all the biopsied embryos were characterized as normal for sex chromosome, the results showed that IMSI results in a significantly higher incidence of female embryos as compared with ICSI (65.1% versus 54.0%, respectively, P=0.0277). After analysing only euploid embryos for the eight selected chromosomes, a significantly higher incidence of XX embryos derived from IMSI was also observed compared with ICSI cycles (66.9% versus 52.5%, respectively, P=0.0322). This result was confirmed by logistic regression, which demonstrated a nearly 2-fold increase in euploid XX embryos derived from spermatozoa selected by high magnification (OR 1.83, 95% CI 1.05-3.35, P=0.032). A higher proportion of morphologically normal spermatozoa analysed under high magnification seem to carry the X chromosome. The aim of this study was to determine if the use of intracytoplasmic morphologically selected sperm injection (IMSI) is associated with gender incidence. Couples who underwent IVF with preimplantation genetic screening, as a result of advanced maternal age, were randomly allocated into two groups: intracytoplasmic sperm injection (ICSI; n=80) or intracytoplasmic morphologically selected sperm injection (IMSI; n=80). The incidences of genders were compared between ICSI- and IMSI-derived embryos. Our results showed that a significantly higher incidence of female embryos derived from IMSI compared with ICSI cycles (66.9% versus 52.5%, respectively, P=0.0322). This result was confirmed by logistic regression, which demonstrated a nearly 2-fold increase in female embryos derived from sperm selected by high magnification (OR 1.83, 95% CI 1.05-3.35, P=0.032). A higher proportion of morphologically normal spermatozoa analysed under high magnification seem to carry the X chromosome.

Varicocelectomy does not impact pregnancy outcomes following intracytoplasmic sperm injection procedures.

There are many studies in the literature suggesting an acquired, apparently progressive infertility due to varicocele. In fact, varicocelectomy has become the most commonly performed male infertility surgery. Assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) are also important for couples with male factor infertility associated with varicocele. Therefore, the aim of this study was to evaluate the effect of varicocelectomy on sperm quality and pregnancy rate with ICSI. Data were analyzed from 248 patients who had varicocele or underwent a previous varicocelectomy and were treated with ICSI between 2000 and 2008. Patients with varicocele were divided into two groups: men with clinical varicocele (group 1, n = 79) and men who underwent varicocelectomy before ICSI (group 2, n = 169). In all cases, female infertility was not detected. We evaluated and compared the two groups' semen characteristics as defined by the World Health Organization and Tygerberg's strict criteria: the female partner's age; the number of oocytes retrieved; and the fertilization, implantation, pregnancy, and miscarriage rates. We used the Wilcoxon signed rank test or the Mann-Whitney test for these analyses. No differences were detected in the age of the female partners between group 1 (33.0 ± 0.46 years) and group 2 (33.8 ± 0.38 years; P = .1872). Semen volume was higher in group 1 (3.3 ± 0.3 mL) than it was in group 2 (2.5 ± 0.14; P = .0043). No differences were detected between groups 1 and 2 with regard to sperm concentration (30.08 ± 4.01 million/mL and 24.1 ± 2.42 million/mL, respectively; P = .138), sperm motility (38.2% ± 2.69% and 38.7% ± 2.08%, respectively; P = .881), and morphology according to Tygerberg's strict criteria (2.6% ± 0.44% and 2.4% ± 0.37%, respectively; P = .7202). Also, no differences were detected in the number of oocytes retrieved between group 1 (14.8 ± 1.74) and group 2 (14.9 ± 1.04; P = .9515). The fertilization rate was higher in group 1 (73.2%) than it was in group 2 (64.9%; P = .0377); however, no differences were detected in the pregnancy rates (31.1% vs 30.9%; P = .9806), implantation rates (22.1% vs 17.3%; P = .5882), or miscarriage rates (21.7% vs 23.9%; P = .8401) between groups 1 and 2. Although a varicocelectomy should always be performed before assisted reproduction is pursued, this surgery does not increase pregnancy rates or decrease miscarriage rates following ICSI.

Prognostic value of triploid zygotes on intracytoplasmic sperm injection outcomes.

PURPOSE: To evaluate the prognostic significance of triploidy incidence on the outcomes of embryos derived from normally fertilized oocytes from the same cohort. METHODS: This study included 1500 ICSI cycles. Logistic regression models were used to study the influence of abnormal fertilization on the development and clinical outcomes of embryos derived from normally fertilized oocytes from the same cohort RESULTS: We observed a negative influence of the percentages of triploid zygotes on fertilization (75.2% and 56.8%, P < 0.0001), high-quality embryos (58.9% and 48.2%, P = 0.0001), pregnancy (34.1% and 28.2%, P = 0.0540) and implantation rates (20.0% and 13.3%, P = 0.0012). When the 3PN zygote rate was >25%, the percentages of normal fertilization, high-quality embryos and implantation rates were significantly lower than in the control group. CONCLUSIONS: We observed an approximately 50% lower risk of pregnancy and a 3.5-fold higher risk of miscarriage in cycles with a 3PN incidence of >25%.

Relationship between oocyte abnormal morphology and intracytoplasmic sperm injection outcomes: a meta-analysis.

The aim of this study was to perform a meta-analysis of the potential effects of oocyte morphological abnormalities on ICSI outcomes. Relevant original papers reporting on the relation between oocyte morphology and ICSI outcomes were identified by searching MEDLINE, EMBASE and the Cochrane Library. The main outcome measures were fertilisation rate and embryo quality. A meta-analysis was performed and Mantel-Haenszel pooled odd ratios (ORs) with 95% confidence intervals (CIs) were calculated to express the relation between the oocyte morphology and the ICSI outcomes. A total of 14 studies reporting 3688 ICSI cycles were included. Our meta-analysis demonstrates that the probability of an oocyte becoming fertilised is significantly reduced by the presence of large IPB (OR: 0.29, CI: 0.09-0.90), large PVS (OR: 0.86, CI: 0.74-0.99), refractile bodies (OR: 0.66, CI: 0.51-0.84) or vacuoles (OR: 0.59, CI: 0.42-0.83). No other investigated morphological abnormalities demonstrated significant relationships with ICSI outcomes. Our data demonstrate that the presence of large IPB, large PVS, refractile bodies or vacuoles is associated with decreased oocyte fertilisation. Our findings might be of importance for selecting embryos for replacement because the effects of oocyte abnormalities on implantation and pregnancy rates remain unclear.

Sperm organelle morphologic abnormalities: contributing factors and effects on intracytoplasmic sperm injection cycles outcomes.

OBJECTIVE: To (1) analyze possible relationships between motile sperm organelle morphology examination (MSOME) and sperm chromatin status, aneuploidy incidence, and patient's age; (2) determine the effects of sperm morphologic abnormalities on intracytoplasmic sperm injection (ICSI) outcomes; and (3) identify the benefits of intracytoplasmic morphologically selected sperm injection (IMSI) in patients with high DNA fragmentation rate. METHODS: The study was performed in 50 patients undergoing ICSI cycles. The MSOME, sperm DNA fragmentation, and sperm aneuploidy incidence were performed in 200 sperm cells of each patient. Regression models were used to assess the relationships among sperm morphology and sperm aneuploidy, sperm DNA fragmentation, patient's age, and ICSI outcomes. In cycles with patients showing a high incidence of DNA fragmentation, oocytes were split into 2 groups according to the sperm selection method: Standard-ICSI (n = 82) and IMSI (n = 79). Fertilization and high-quality embryo rates were compared between the groups. RESULTS: A close relationship between sperm DNA fragmentation and the presence of vacuoles in the MSOME was noted. The patient's age was correlated to the presence of vacuoles. No correlation between sperm aneuploidy and IMSI was observed. Vacuolated cells were negatively correlated with fertilization, pregnancy, and implantation. In patients with a high incidence of sperm DNA fragmentation, fertilization and high-quality embryo rates were similar when comparing IMSI and Standard-ICSI. CONCLUSIONS: Our data demonstrate a correlation between paternal age and the incidence of nuclear vacuoles, as well as an effect of large and small vacuoles on late embryo development.

Secretome of the preimplantation human embryo by bottom-up label-free proteomics.

A bottom-up label-free mass spectrometric proteomic strategy was used to analyse the protein profiles of the human embryonic secretome. Culture media samples used for embryonic culture of patients undergoing intracytoplasmic sperm injection cycles were selected as a test case for this exploratory proof-of-principle study. The media were stored after embryo transfer and then pooled into positive (n = 8) and negative (n = 8) implantation groups. The absolute quantitative bottom-up technique employed a multidimensional protein identification technology based on separation by nano-ultra-high pressure chromatography and identification via tandem nano-electrospray ionization mass spectrometry with data-independent scanning in a hydrid QqTOF mass spectrometer. By applying quantitative bottom-up proteomics, unique proteins were found exclusively in both the positive- and negative-implantation groups, which suggest that competent embryos express and secrete unique biomarker proteins into the surrounding culture medium. The selective monitoring of these possible secretome biomarkers could make viable procedures using single-embryo transfer.

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential.

BACKGROUND: The metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues). METHODS: We analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel). RESULTS: The results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples. CONCLUSION: Our results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient.