Clinical outcomes following ICSI cycles using surgically recovered sperm and the impact of maternal age: 2004-2015 SART CORS registry.

PURPOSE: The aims of this study were (1) to evaluate clinical outcomes after ICSI cycles using surgically recovered sperm and (2) to assess the influence of maternal age on those outcomes. METHODS: A retrospective cohort study of 24,763 IVF cycles of fresh autologous oocytes and ICSI using surgically recovered sperm reported to the SART CORS database from 2004 to 2015. RESULTS AND CONCLUSIONS: Older women had significantly longer stimulation (p < 0.001), a lower number of oocytes retrieved (p < 0.001), a lower number of 2PN zygotes (p < 0.001), a lower chance of having a blastocyst transferred (p < 0.001), and a higher number of fresh embryos transferred (p < 0.001). There was no significant association between the number of 2PNs per oocyte retrieved and maternal age (p = 0.214). Both clinical pregnancy rates and live birth rates (LBR) decreased with advanced maternal age (p < 0.001). LBR ranged from 50.4% in women < 30 to 7.2% in women > 42 years, and for cleavage-stage transfers, the LBR ranged from 47.3% in women< 30 to 6.3% in women > 42 years. There were no differences in gestational age at delivery, proportion of term deliveries, preterm deliveries, neonatal birth weight < 2500 g, neonatal birth weight > 4000 g and average birthweight of neonates for singleton pregnancies according to age. For twin pregnancies, women < 30 years had significantly higher number of live births, term deliveries, and lower preterm deliveries than older women. There was a similar number of female (6051) and male neonates (5858; p = 0.2). Overall, pregnancy outcomes with ICSI using surgically recovered sperm are reassuring and comparable to those of ICSI with ejaculated sperm.

ICSI outcomes in men undergoing TESE for azoospermia and impact of maternal age.

This retrospective study compared clinical outcomes in men with obstructive and nonobstructive azoospermia after ICSI following testicular sperm extraction and the influence of maternal age. Fertilisation rates, embryo quality, pregnancy rates, miscarriage rates and live birth rates were evaluated. Men with obstructive azoospermia (OA) had significantly higher rates of diploid fertilisation and clinical pregnancy than men with nonobstructive azoospermia (NOA), but miscarriage rates and live birth rates were not significantly different. The higher rates of fertilisation, embryo quality and clinical pregnancy in men with OA were statistically significant when their female partners were <35 years but results were similar in both groups when female partners ≥35 years. Although the OA group had better overall quality embryos than the NOA group when maternal age was <35 years, embryologists can select the morphologically better embryos for transfer, eliminating the effect of embryo quality differences present in these two groups. Understanding more about factors that affect TESE/ICSI outcomes will not only help us predict patients' outcomes but it can help us educate and better counsel our patients.

Transcriptome of early embryonic invasion at implantation sites in a murine model.

Successful implantation relies on the interaction between a competent embryo and a receptive endometrium. The aim of the present study was to investigate genes differentially expressed in early invasive embryonic tissue versus decidual tissue in mice. Samples were obtained from the ectoplacental cone, the immediately surrounding deciduas and from deciduas from interimplantation sites. Microarray analysis showed that 817 genes were differentially expressed between extra-embryonic tissue and the surrounding decidua and that 360 genes were differentially expressed between the different deciduas, with a high representation of developmental processes. Genes differentially expressed in the maternal compartment included chemokines, lipoproteins, growth factors and transcription factors, whereas the embryonic invasive tissue expressed genes commonly observed in invasive tumour-like processes. These results provide information about genes involved in early embryonic invasion and the control exerted by the surrounding decidua. This information may be useful to find targets involved in pathologies associated with implantation failure and early pregnancy loss.

Milk fat globule epidermal growth factor 8 (MFG-E8): a novel protein in the mammalian endometrium with putative roles in implantation and placentation.

OBJECTIVES: MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites. STUDY DESIGN: MFG-E8 protein and its receptor integrin αvß3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR. MAIN OUTCOME MEASURES: Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites. RESULTS: MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age. CONCLUSIONS: MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.

Clinical management of male infertility in assisted reproduction: ICSI and beyond.

The advent of in vitro fertilization and its augmentation with intracytoplasmic sperm injection (ICSI) has allowed a large number of couples suffering from moderate to severe male infertility, and also presenting with female pathologies, to achieve their reproductive dreams. Notwithstanding the existence of fundamental questions about the pathophysiological mechanisms leading to sperm dysfunction, and still unanswered concerns about health risks following ICSI, it appears that overall ICSI is safe and here to stay. Although on one hand ICSI possibly hampered advances of the knowledge in some areas of gamete biology and interaction, on the other it definitely gave impulse to studies designed to unveil the sperm contributions during and beyond fertilization, including the normalcy of the DNA/chromatin as well as molecular mechanisms of genetic/epigenetic control and nuclear organization status. In all, almost entering the fourth decade of assisted reproductive technologies, we should continue monitoring the safety of the technique and long-term development of offspring, whereas at the same time prioritizing areas of research addressing these fundamental questions.

Ovulation induction in IVF.

This review examined current controlled ovarian hyperstimulation (COH) protocols used in ART. Controversies still exist regarding selection of gonadotropin preparation (i.e., recombinant versus urinary forms, pure FSH versus FSH-LH containing preparations), choice of adjuvant therapy with GnRH analogues (agonists versus antagonists), and pretreatment with oral contraceptive pills. Patients prospectively identified as intermediate responders have excellent outcomes with adjuvant therapy with either a GnRH agonist (long protocol) or a GnRH antagonist, but tailoring of gonadotropin dose and type must be performed to achieve optimized results. High responders perform satisfactorily with gentler gonadotropin stimulation regimens that minimize the occurrence of ovarian hyperstimulation syndrome. On the other hand, results in low/poor responders remain sub optimal both in terms of ovarian response and oocyte/embryo quality in spite of a variety of stimulation approaches and adjuvant therapies implemented. It is concluded that ovarian stimulation is a critical step in IVF therapy. There are a variety of available and efficacious novel COH regimens but individualization of management is essential and dependent upon proper and prospective assessment of the ovarian reserve. The identification of the pathogeneses underlying poor ovarian response constitutes a formidable challenge facing reproductive endocrinologists.

Poor responders in in vitro fertilization (IVF) therapy: the challenge continues.

Poor responders represent more than a third of women undergoing assisted reproduction. Typically they are patients with advanced maternal age and low ovarian reserve. However, there is a younger group that unexpectedly demonstrates impaired response to controlled ovarian hyperstimulation. The etiologies in many of these cases are still unclear. In our program, the determination of basal cycle day 3 serum FSH, LH and E2 levels, measurement of AMH, and the estimation of the basal antral follicular count by transvaginal ultrasonography, are the preferred screening tests for ovarian reserve in all IVF patients, and together with the woman's age, determine the ovarian stimulation regimen to be chosen for the cycle treatment. In spite of a -variety of protocols and adjuvant therapies of unproven benefit, these patients have compromised outcomes and continue to represent a challenge to reproductive endocrinologists.

Impact of male age on the outcome of assisted reproductive technology cycles using donor oocytes.

This study assessed the influence of the age of the male partner on the outcome of oocyte donation cycles. A total of 408 couples participating in 519 consecutive anonymous oocyte donation cycles were examined. Main outcome measures were fertilization rate, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates, as well as the total reproductive potential, which estimates the outcome from fresh and cryopreserved-thawed embryo transfers. A total of 241 cycles resulted in clinical pregnancy (48.5% of transfers). The mean embryo score for transferred embryos (ESTE) was higher in cycles resulting in pregnancy (P=0.003). Semen volume (P<0.001), sperm motility (P<0.001) and fertilization rate (P=0.04) decreased significantly with advanced male age, which did not correlate with mean ESTE or implantation rate. Fertilization rate was the only predictor of ESTE (B=16.066, P=0.012), whereas inseminated/retrieved egg ratio was the only predictor of implantation rate (B=0.555, P=0.039). Pregnancy was only predicted by ESTE (Exp(B)=1.023, P<0.001), which also was the only predictor of live birth (Exp(B)=1.017, P=0.009). There was no predictor of miscarriage (47 cycles, 9.1%) identified. Although semen volume, sperm motility and fertilization rate decreased with advanced male age, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates were not affected.

Complete globozoospermia associated with PLCζ deficiency treated with calcium ionophore and ICSI results in pregnancy.

Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCzeta), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCzeta was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.

Oligozoospermia and heat-shock protein expression in ejaculated spermatozoa.

BACKGROUND: Heat-shock protein A2 (HspA2) is correlated with sperm maturity, function and fertility, and a dysfunctional expression of such a gene results in abnormal spermatogenesis. The purpose of this study was to compare HspA2 gene expression in spermatozoa from oligozoospermic men and normozoospermic controls. METHODS: Semen was obtained and analysed according to World Health Organization (World Health Organization, 1999) guidelines, morphology by Kruger's strict criteria. Seventeen patients with oligozoospermia and 21 fertile controls were studied. Total RNA was extracted from ejaculated and Percoll density-gradient-separated spermatozoa followed by semiquantitative RT-PCR analysis. The relative expression level of HspA2 was analysed according to the expression level of the housekeeping beta-actin gene. Serum hormonal profiles (FSH, LH and testosterone) and a peripheral karyotype were also performed. RESULTS: All patients possessed normal karyotype, and no significant hormonal differences were found between the two groups. The study group had significantly lower sperm concentration and normal morphology than the controls. Semiquantitative RT-PCR analysis of HspA2 showed significantly lower expression levels in the oligoteratozoospermic men when compared to controls (P=0.0021). CONCLUSIONS: The HspA2 gene was down-regulated in sperm from infertile men with idiopathic oligoteratozoospermia, suggesting that such anomalies of gene expression might be associated with pathogenesis in some subtypes of male infertility.

In search of candidate genes critically expressed in the human endometrium during the window of implantation.

BACKGROUND: In this prospective randomized blinded clinical trial, we examined gene expression profiles of the human endometrium during the early and mid-luteal phases of the natural cycle. METHODS: An endometrial biopsy was performed on day 16 (LH +3) or on day 21 (LH +8), followed by RNA extraction and microarray analysis using an Affymetrix HG-U95A microchip. Data analysis was carried out using pairwise multiple group comparison with the significance analysis of microarrays (SAM) software. RESULTS: With a false discovery rate of 0, the analysis revealed that 107 genes were significantly and differently expressed (> or =2-fold) during the early versus the mid-luteal phase of the cycle. Forty-five of these genes have not been previously linked to endometrial receptivity. Validation of the microarray data was accomplished using semiquantitative RT-PCR. We demonstrated the presence of estrogen and progesterone response elements (ERE and PRE) by analysis of the 5'-flanking regions of a subset of differentially regulated genes. CONCLUSIONS: Using a strict bioinformatics approach of microarray data, we demonstrated significant changes in candidate genes during the transition of the early to the mid-luteal phase of the human endometrium that may have functional significance for the opening and maintenance of the window of implantation.

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa.

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm-oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm-egg interaction in bovine and other species.

The use of semen parameters to identify the subfertile male in the general population.

AIMS: To present a structured review of the literature published on semen parameters and in vivo fertility potential and to establish fertility/subfertility thresholds for sperm morphology using Tygerberg strict criteria, sperm concentration, and sperm motility. METHOD: The published literature comparing fertile and subfertile populations between 1983 and 2002 was reviewed. RESULTS: A total of 265 articles were identified by the sourcing methodology, but only four articles provided data that could be tabulated and analyzed. Using receiver-operating characteristics curves, morphology proved to be the best predictor of subfertility in 2 of the 4 articles, with concentration and motility also showing good predictive power. The thresholds calculated ranged between 4 and 10% for morphology, between 13.5 x 10(6)/ml and 34 x 10(6)/ml for concentration, and between 32 and 52% for motility. A second set of much lower thresholds was calculated in three of the articles using either a 15 or 50% prevalence of subfertility in the population or the tenth percentile of the fertile population. The adjusted thresholds were between 3 and 5% for morphology, between 9 x 10(6)/ml and 20 x 10(6)/ml for concentration, and between 20 and 30% for motility. CONCLUSIONS: Because these lower thresholds have a much higher positive predictive value, we suggest that thresholds of <5% normal sperm morphology, a concentration <15 x 10(6)/ml, and a motility <30% should be used to identify the subfertile male. The lower threshold for morphology also fits in vitro fertilization and intrauterine insemination data calculated previously. Using the parameters in combination increases the clinical value of semen analysis.

Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality.

In this study we extended earlier work to determine whether sperm respond to somatic cell apoptotic stimuli and whether apoptotic phenotypes are significant indicators of human sperm quality. We evaluated ejaculated sperm from fertile donors and subfertile patients following purification of fractions of high and low motility. In unstimulated conditions, caspase enzymatic activity was higher in motile fractions from subfertile patients than in donors, and was higher in low motility fractions from both groups. Staurosporine, but not a Fas ligand or H2O2, significantly increased caspase activity, but only in high motility fractions. Procaspase-3, -7 and -9 and low levels of active caspase-3, -7 and -9 were identified by immunoblot analysis. Apoptosis-inducing factor (AIF) was present in all samples but poly ADP-ribose polymerase-1 (PARP-1) was not detected. Phosphatidylserine translocation was significantly increased only with H2O2 treatment. In ejaculates of both subfertile and fertile men, we demonstrated the presence and activation of several proteins that are key constituents of apoptosis-related pathways in somatic cells, which may serve as markers for sperm quality.

Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study.

BACKGROUND: We aimed to investigate whether sperm DNA quality may predict intrauterine insemination (IUI) outcome. METHODS: The study was designed in a prospective cohort fashion, at a tertiary centre for reproductive medicine. A total of 119 patients underwent 154 cycles of IUI. Parameters related to demography, cycle management and semen sample used for IUI were evaluated. Conventional semen parameters, morphology (strict criteria), sperm DNA fragmentation and stability [evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and acridine orange staining under both acid and acid + heat denaturing conditions respectively] were measured. The main outcome measure was clinical pregnancy, defined as ultrasonographic visualization of intrauterine gestational sac(s). RESULTS: Logistic regression analyses were done on six sets of data, including all cycles combined, cycles with washed samples, first cycle of each couple, first cycle of each couple with washed samples, cycles stimulated with gonadotrophins and finally gonadotrophin-stimulated cycles with washed samples. The number of pre-ovulatory follicles on day of hCG, the age of the woman and the percentage of sperm with acid- + heat-resistant DNA were the parameters that predicted IUI outcome in most of these data subsets. For the gonadotrophin-stimulated cycles, age of the man appeared as a predictor as opposed to that of the woman; and for the cycles within this subgroup, where the semen sample was washed, sperm DNA fragmentation and age of the man were the only two parameters to predict IUI outcome. No samples with >12% of sperm having DNA fragmentation resulted in pregnancy. CONCLUSIONS: The number of follicles, age of the woman/man and sperm DNA quality may predict IUI outcome.

Zona pellucida induced acrosome reaction, sperm morphology, and sperm-zona binding assessments among subfertile men.

PURPOSE: The study aimed to evaluate the relationship between the zona pellucida induced acrosome reaction (ZIAR) and (i) percentage normal spermatozoa as well as (ii) sperm-zona pellucida binding potential among men referred for a routine semen analysis. METHODS: Semen samples of 164 consecutive men referred to the andrology laboratory for routine semen analysis were studied. Semen samples were analyzed using the new WHO standards (strict criteria). ZIAR was recorded with a lectin conjugated Pisum sativum agglutinin microassay, while sperm-zona binding was evaluated with a standard hemizona assay (HZA). RESULTS: Andrology patients were divided according to the percentage normal spermatozoa in the ejaculate, namely <4% normal forms (n = 71), 5-14%, normal forms (n = 73), and >14% normal forms (n = 20). ZIAR data of the <4%, 5-14%, and >14% groups was (9.6 +/- 0.6)%, (13.9 +/- 0.5)%, and (15.0 +/- 1.1)%, respectively. The ZIAR data of fertile control men was (26.6 +/- 1.4)% which differed significantly from the three andrology referrals groups. Likewise significant differences were recorded during the hemizona assay namely, 38.0% (<4% normal forms), 54.5% (5-1% normal forms), and 62.6% (>14% normal forms). Among the group with >14% normal forms, five cases had impaired ZIAR outcome (<15%). Three of these men had normal morphology and HZAs. CONCLUSIONS: ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of andrology referrals revealed an impaired acrosome reaction response to solubilized zona pellucida.

Delaying the initiation of progesterone supplementation results in decreased pregnancy rates after in vitro fertilization: a randomized, prospective study.

OBJECTIVE: To compare IVF outcome between two protocols for luteal phase supplementation, one beginning on day 3 after oocyte retrieval and the other beginning on day 6 after retrieval. DESIGN: Prospective, randomized study. SETTING: University-based assisted reproductive technology center. PATIENT(S): One hundred twenty-six consecutive patients undergoing IVF between January and July 2000. INTERVENTION(S): Patients were randomized to begin luteal phase support using vaginal progesterone beginning either on day 3 after oocyte retrieval or on day 6 after oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates and implantation rates. RESULT(S): All patients randomized underwent transfer. There were no differences in age, oocytes retrieved, or embryos transferred between the two groups. Those patients receiving luteal phase support with progesterone beginning on day 6 after retrieval had a significantly lower clinical pregnancy rate per transfer compared with those beginning support on day 3 after retrieval (44.8% vs. 61.0%, respectively). This difference in pregnancy rates was greater in those patients undergoing a luteal gonadotropin releasing hormone (GnRH) agonist down-regulation protocol (47.5% vs. 71.4%, day 6 vs. day 3, respectively). Beginning support on day 6 also significantly decreased implantation rates in the GnRH agonist group (21.0% vs. 34.0%, day 6 vs. day 3, respectively). CONCLUSION(S): Pregnancy rates are significantly decreased by initiating luteal-phase progesterone supplementation on day 6 after oocyte retrieval during in vitro fertilization cycles.

Strategies for the infertile man.

Male infertility is one of the most common, identifiable causes of human reproductive failure. Although considerable progress has been made toward understanding sperm physiology and the biology of gamete interaction, still more work is needed to achieve objectivity and standardization of some of the andrological diagnostic methods used in the clinical setting. More information is needed to definitively establish which tests are more accurate predictors of sperm performance and how they correlate with pregnancy potential following in vivo and in vitro interventions. Infertile men can be successfully treated with defined urological and medical therapies or with assisted reproductive technologies (ARTs). Among the latter, intracytoplasmic sperm injection (ICSI) has become a validated means to overcome multiple sperm deficiencies. Nevertheless, it is expected that simplified and more cost-efficient therapeutic modalities will be developed as additional basic (cellular-molecular) and clinical knowledge is gained.