Algorithm-based individualization methodology of the starting gonadotropin dose in IVF/ICSI and the freeze-all strategy prevent OHSS equally in normal responders: a systematic review and network meta-analysis of the evidence.

PURPOSE: Ovarian hyperstimulation syndrome (OHSS) represents a rare but dangerous condition associated with controlled ovarian stimulation (COS) in IVF/ICSI. Over the last decades, many strategies have been introduced into clinical practice with the objective of preventing this potentially life-threatening condition. Among these, the freeze-all policy has gained great popularity, thanks to improvements in vitrification. Nevertheless, not all clinics have adequate skills in vitrification procedures and patients may be dissatisfied with a longer time to pregnancy. METHODS: This study is a systematic review and network meta-analysis of randomized controlled trials comparing different strategies of ovarian stimulation in IVF/ICSI cycles (freeze-all policy, algorithm-based individualization of the starting dose, experience-based individualization of the starting dose, standard dose) in terms of reduction of OHSS, in normal responders. RESULTS: The results indicate that only the algorithm-based individualization of the starting gonadotropin dose reduces OHSS similarly to the freeze-all strategy. CONCLUSION: Albeit in the era of the freeze-all policy, the personalization of the starting gonadotropin dose obtained by the use of algorithms should be pursued as a valid and safe option for IVF.

A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles.

The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8-14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

The carriers of the A/G-G/G allelic combination of the c.2039 A>G and c.-29 G>A FSH receptor polymorphisms retrieve the highest number of oocytes in IVF/ICSI cycles.

PURPOSE: The objective of this study was the elucidation of the possible role of the single-nucleotide polymorphisms (SNP) at position -29 and 2039 of the FSH receptor gene (FSHR) as independent predictive markers of ovarian response. Indeed, the tailoring of reproductive treatments is crucial for both maximizing the success of IVF patients and obtaining a reduction in hypo- or hyper-response rates. METHODS: This prospective, observational study analyzed the association of -29 and 2039 FSHR polymorphisms with the number of retrieved oocytes in 140 patients attending an IVF/ICSI cycle for severe male factors (≤5,000,000 spermatozoa/mL) or tubal factors at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULTS: The results of this study demonstrate that the genetic combination of A/G for polymorphism c.2039 A>G with G/G for polymorphism c.-29 G>A is significantly associated with the highest number of collected oocytes (p = 0.03). This association was significant even after controlling for the effect of other clinical variables. CONCLUSIONS: The A/G-G/G allelic variant, identified as an independent variable, if confirmed in a larger number of patients, could be considered as a new genetic biomarker, which could increase the efficacy of prediction models for ovarian stimulation.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

OBJECTIVE: The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. METHOD: Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin. RESULTS: Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. CONCLUSIONS: The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John Wiley & Sons, Ltd.

The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures.

PURPOSE: The aim of this study was to investigate the most suitable sperm preparation technique to apply in order to obtain a spermatozoon population with minimal DNA damage during in vitro fertilization procedures. We compared four preparation techniques: direct swim-up (DSU), pellet swim-up (PSU), density gradient (DG), and density gradient followed by swim-up (DG-SU), evaluating the effects of each technique on the DNA damage rate, evaluated by DNA fragmentation index of the spermatozoa obtained. METHODS: In this observational study, 98 semen samples from couples undergoing IVF/ICSI cycles were included. Data were collected between April and November 2014 at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULT(S): The percentages of DNA fragmentation were 18.30 ± 10.8 in raw samples, 6.6 ± 5.7 after DSU, 4.2 ± 3.8 after PSU, 12.9 ± 9.9 after DG, and 3.7 ± 4.0 after DG-SU respectively. Compared to the raw evaluation, all the preparation techniques significantly decreased the total rate of the DNA fragmentation (DSU Z = -8.60, P < 0.008; PSU Z = -8.54, P < 0.008; DG Z = -6.42, P < 0.008, and DG-SU Z = -8.60, P < 0.008, respectively). Comparing them, spermatozoa with intact DNA after PSU and DG-SU were significantly higher than after DSU (Z = -7.12, P < 0.008; Z = -6.59, P < 0.008, respectively) and after DG (Z = -8.41, P < 0.008; Z = -8.60, P < 0.008, respectively). The difference between PSU and DG-SU was not significant (Z = -2.21, P = 0.03). CONCLUSION(S): There are, above all, two techniques of sperm preparation which allow for the recovery of spermatozoa with the lowest DNA fragmentation rate. Furthermore, given low costs and reduced time, we believe that PSU is the best option in the treatment of semen samples during IVF/ICSI.

The gene expression profile of cumulus cells reveals altered pathways in patients with endometriosis.

PURPOSE: The objective of this experimental study was to compare the global gene expression profile of CC of mature oocytes in 18 patients with severe endometriosis and CC in 18 control patients affected by a severe male factor. METHODS: For each group, the CC were pooled, RNA was extracted and a microarray performed. For validating the microarray, a quantitative real-time PCR was performed in the CC of an independent set of patients with endometriosis (n = 5) and controls (n = 7). RESULTS: 595 differentially expressed genes (320 down-regulated, 275 up-regulated, p < 0.05, fold change ≥1.5) were identified. The most significant changes were observed in genes involved in the chemokine signaling and cell-cell or cell-extracellular matrix adhesion pathways. Several genes of these pathways were down-regulated in endometriosis. Individual RT-PCR assays confirmed the microarray for ten genes. CONCLUSIONS: Several genes involved in the chemokine mediated-signaling pathway and in the functional cross-talk between CC and the oocyte are down-regulated in endometriosis CC. The impairment of these processes could explain the reduction of oocyte competence in endometriosis. This preliminary knowledge could be the starting point for a more detailed elucidation of the relationship between endometriosis and oocyte competence.

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

The paradox of the Italian clinical embryologist in the national public health system: hints towards harmonization of a postgraduate educational curriculum.

Clinical embryologists are highly trained laboratory professionals with multiple roles, including laboratory, clinical, biobanking and quality system management. In most European countries, clinical embryologists are trained to work in Medically Assisted Reproduction (MAR) centres without a specifically dedicated educational path. The criteria required for employment vary according to the educational structure and the public or private nature of the centre. We have herein described the educational profile required by Italian clinical embryologists to work in MAR centres of the National Health System (NHS). Public centres currently represent 36% of all the Italian MAR clinics. According to the Italian law, a future clinical embryologist must achieve a 3-4 year unpaid post-graduate specialization in a different field, choosing from Genetics, Microbiology, Clinical Pathology or Nutrition. Accesses to the above-mentioned post-graduate courses are themselves very limited. Clinical embryologists are basically trained by senior colleagues. This situation makes inevitably difficult to recruit laboratory staff in NHS centres. Moreover, it represents an emblematic example of the need for an equal training curriculum, possibly ensuring a comparable education quality, mobility of trainees and dissemination of skills for clinical embryologists all over Europe.

Infertility-related stress, anxiety and ovarian stimulation: can couples be reassured about the effects of psychological factors on biological responses to assisted reproductive technology?

The aim of this prospective, longitudinal study was to examine the association between couples' pre-treatment psychological characteristics (state anxiety and infertility-related stress levels of both partners) and ovarian response during assisted reproductive technology treatment in a well-controlled sample. A total of 217 heterosexual couples (434 patients), suffering from primary infertility and undergoing their first assisted reproductive technology treatment at the Reproductive Medicine Unit of ANDROS Day Surgery Clinic in Palermo (Italy), were recruited. Psychological variables were assessed using the State Scale of State-Trait Anxiety Inventory (STAI-S) and the Fertility Problem Inventory (FPI). The number of follicles ≥ 16 mm in diameter, evaluated by transvaginal ultrasound scan on the eleventh day of the workup, was chosen as the outcome measure. No association between women's level of anxiety and infertility-related stress, and the number of follicles ≥ 16 mm in diameter was found. Moreover, the male partner's infertility stress and anxiety did not influence the relationship between the woman's infertility-related stress, anxiety level and ovarian response. Fertility staff should reassure couples that the woman's biological response to ovarian stimulation is not influenced by either partner's level of psychological distress.

Assessing infertility-related stress: the factor structure of the Fertility Problem Inventory in Italian couples undergoing infertility treatment.

The factor structure of the Fertility Problem Inventory (FPI) and its invariance across gender were examined in Italian couples undergoing infertility treatment. About 1000 subjects (both partners of 500 couples) completed two questionnaires prior to commencing infertility treatment at a private Clinic in Palermo, Italy. Confirmatory Factor Analysis demonstrated that the original factor structure of the FPI was partially confirmed. Two correlated factors (Infertility Life Domains and Importance of Parenthood) were obtained via a post hoc Exploratory Factor Analysis. Finally, the invariance of this factor structure across gender was confirmed. The study supported the relevance of two interrelated factors specific to infertility stress which could help clinicians to focus on the core infertility-related stress domains of infertile couples.

Number of good quality embryos on day 3 is predictive for both pregnancy and implantation rates in in vitro fertilization/intracytoplasmic sperm injection cycles.

OBJECTIVE: To evaluate the predictive value for implantation and pregnancy rates of the number of embryos that reach the eight-cell stage with less than 20% fragmentation (good quality embryos) on day 3. DESIGN: Prospective observational study. SETTING: Private IVF center. PATIENT(S): One hundred eighty-nine women undergoing IVF, or intracytoplasmic sperm injection with at least four zygotes, who have had three embryos transferred on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate and implantation rate. RESULT(S): All patients (n = 189) had 3 embryos transferred. The patients were divided in five groups according to the number of good quality embryos on day 3 (0, 1, 2, 3, >3; respectively). For the five groups, the pregnancy rates were 2.9%, 33.3%, 45.5%, 39.4%, and 64.3%; the implantation rates were 2.0%, 17.0%, 20.5%, 19.3%, and 35.7%. No statistically significant differences in pregnancy rate or implantation rate were found for groups 1, 2, and 3 (when 1, 2, or 3 good quality embryos were present). The data were also analyzed after pooling these three groups. A statistically significant difference in pregnancy rate and implantation rate was found for group 0 (no good quality embryos), groups 1, 2, and 3 (1, 2, or 3 good quality embryos), and group >3 (>3 good quality embryos). CONCLUSION(S): The number of good quality embryos available on day 3 is a strong predictive value for both pregnancy rate and implantation rate. When good quality embryos are present on day 3, only two embryos should be transferred to minimize multiple pregnancies.

A "four-cycle program" improves the estimate of the cumulative pregnancy rate and increases the number of actual pregnancies in IUI treatment: a cohort study.

OBJECTIVE: To demonstrate that reduction of the cumulative dropout rate (CDR) improves the accuracy of the estimate of the cumulative pregnancy rate (CPR) in a set of four intrauterine insemination (IUI) cycles ("four-cycle program") and increases the total number of pregnancies obtained. STUDY DESIGN: Single-centre retrospective observational cohort study of couples who underwent IUI cycles at the Andros Day Surgery Clinic, Palermo, from 1997 to 2011. The main outcome measure was the calculation of the CPR, with life table analysis, firstly by giving the same probability of pregnancy to the dropouts as the patients who continued the treatment (usual method) and secondly by considering this probability null (conservative method). The difference between these two methods was used to verify the accuracy of the estimate. RESULT(S): In the 15 years, 924 couples underwent 2956 cycles carried out consecutively in a set of four cycles. The CDR was 16%. The CPR was 31.4% with the usual method and 29.1% with the conservative method. The difference between the two estimates was not significant, indicating a high reliability of the results and a good accuracy of the calculation. Furthermore, maintenance of a low CDR permits improvement of the CPR, as was demonstrated by considering scenarios with worse dropout rates. CONCLUSION(S): The "four-cycle program" results in a reduction in the CDR, allowing a better estimation of the CPR, and increases the number of actual pregnancies in IUI. The CPR should become the focus for reporting outcome rates in IUI cycles. Reduction of the dropout rate allows us to give the patient a more reliable and accurate estimate of the pregnancy rate.

Conjoined twins in a trichorionic quadruplet pregnancy after intracytoplasmic sperm injection and quarter laser-assisted zona thinning.

OBJECTIVE: To describe the occurrence and management of conjoined twins (CTs) in a trichorionic quadruplet pregnancy after the transfer of three embryos obtained by intracytoplasmic sperm injection (ICSI) that had undergone assisted zona hatching (AZH) using a noncontact laser. DESIGN: Case report. SETTING: Private and public IVF centers. PATIENT(S): A 38-year-old nulliparous woman. INTERVENTION(S): IVF, diagnosis by two-dimensional, three-dimensional, and four-dimensional ultrasound, selective termination at 12 weeks gestation. MAIN OUTCOME MEASURE(S): Clinical follow-up and delivery. RESULT(S): After diagnosis, counselling, and selective termination, the subsequent prenatal course was uneventful, and two healthy boys were delivered by elective cesarean section at 38 weeks gestation. CONCLUSION(S): Monozygotic twinning (MZT) and CTs could complicate multifetal pregnancies obtained by assisted reproduction techniques. The relationship between CTs, IVF, and AZH should be better studied to fully understand the mechanisms that lead to MZT (with the possible role of AZH) and CTs in MZT, in particular. Three-dimensional and four-dimensional ultrasound improve prenatal assessment of the condition. Selective embryo reduction is a safe procedure for the management of these pregnancies.