The Reproductive Journey in the Genomic Era: From Preconception to Childhood.

It is estimated that around 10-15% of the population have problems achieving a pregnancy. Assisted reproduction techniques implemented and enforced by personalized genomic medicine have paved the way for millions of infertile patients to become parents. Nevertheless, having a baby is just the first challenge to overcome in the reproductive journey, the most important is to obtain a healthy baby free of any genetic condition that can be prevented. Prevention of congenital anomalies throughout the lifespan of the patient must be a global health priority. Congenital disorders can be defined as structural or functional anomalies that occur during intrauterine life and can be identified prenatally, at birth, or sometimes may only be detected later during childhood. It is considered a frequent group of disorders, affecting 3-6% of the population, and one of the leading causes of morbidity and mortality. Congenital anomalies can represent up to 30-50% of infant mortality in developed countries. Genetics plays a substantial role in the pathogenesis of congenital anomalies. This becomes especially important in some ethnic communities or populations where the incidence and levels of consanguinity are higher. The impact of genetic disorders during childhood is high, representing 20-30% of all infant deaths and 11.1% of pediatric hospital admissions. With these data, obtaining a precise genetic diagnosis is one of the main aspects of a preventive medicine approach in developed countries. The field of reproductive health has changed dramatically from traditional non-molecular visual microscope-based techniques (i.e., fluorescence in situ hybridization (FISH) or G-banding karyotype), to the latest molecular high-throughput techniques such as next-generation sequencing (NGS). Genome-wide technologies are applied along the different stages of the reproductive health lifecycle from preconception carrier screening and pre-implantation genetic testing, to prenatal and postnatal testing. The aim of this paper is to assess the new horizon opened by technologies such as next-generation sequencing (NGS), in new strategies, as a genomic precision diagnostic tool to understand the mechanisms underlying genetic conditions during the "reproductive journey".

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18 years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.

Clinical application of embryo aneuploidy testing by next-generation sequencing.

We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid embryos. In contrast, the number of MII oocytes retrieved or the use of oocyte vitrification did not affect aneuploidy rates. Similarly, neither maternal age, oocyte number, nor oocyte vitrification affected the incidence of mosaicism. Analysis of clinical outcomes, indications, and potential benefits of embryo aneuploidy testing revealed advanced maternal age as the most favored group, with some evidence of improved delivery rate per transfer as well as decreased miscarriage rates and time to pregnancy. Other indications are: recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and good prognosis patients mainly undergoing single embryo transfer, with the latter indication used to reduce the occurrence of multiple pregnancies without compromising cycle outcome. In conclusion, NGS has become the most appropriate technology for aneuploidy testing in trophectoderm biopsies, with accurate results, high throughput, and cost efficiency. This technology can be also applied to the analysis of the embryonic cell free DNA released to the culture media at blastocyst stage. This is a promising approach towards a non-invasive preimplantation genetic testing of aneuploidy.

Morphokinetics as a predictor of self-correction to diploidy in tripronucleated intracytoplasmic sperm injection-derived human embryos.

OBJECTIVE: To describe, in morphokinetic terms, a tripronucleated embryo (TPN) population according to ploidy and to explore the value of such variables for predicting ploidy. DESIGN: Experimental. SETTING: In vitro fertilization laboratory. PATIENT(S): Seventy-nine TPN embryos obtained after intracytoplasmic sperm injection (TPN-ICSI) were cultured in a time-lapse incubator for 6 days. INTERVENTION(S): Ploidy determinations were carried out for 35 TPN-ICSI at the cleavage and/or blastocyst stage. Their morphokinetics were then retrospectively compared. MAIN OUTCOME MEASURE(S): Direct (cleavage time from 2- to 8-cell stages) and indirect (cell cycle duration and blastomere synchrony at cleavage) morphokinetic variables; ploidy determination by FISH; in vitro development to the blastocyst stage. RESULT(S): TPN-ICSI cleaved later than bipronucleated control embryos (BPN). Diploid TPN displayed morphokinetic behavior closer to BPN than triploid TPN regarding almost all of the direct and indirect morphokinetic variables measured. Variable t5 was found to be a predictable variable of ploidy in TPN. CONCLUSION(S): TPN-ICSI are not homogeneous in ploidy, cleavage, or morphokinetic terms. Diploid, but nontriploid, TPN are morphokinetically similar to diploid BPN. The ploidy of TPN can be predicted by variable t5.

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies.

OBJECTIVES: The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs-on-Beads(TM) (PNBoBs(TM) ) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBs(TM) under different prenatal indications. METHODS: A total of 9648 prenatal samples were prospectively analyzed by karyotyping plus PNBoBs(TM) and classified by prenatal indication. The frequencies of the genomic defects and their 95%CIs were calculated for each indication. RESULTS: The overall incidence of cryptic imbalances was 0.7%. The majority involved the DiGeorge syndrome critical region (DGS). The additional diagnostic yield of PNBoBs(TM) in the population with a low a priori risk was 1/298. The prevalences of DGS microdeletion and microduplication in the low-risk population were 1/992 and 1/850, respectively. CONCLUSIONS: The constant a priori risk for common pathogenic cryptic imbalances detected by this technology is estimated to be ~0.3%. A prevalence higher than that previously estimated was found for the 22q11.2 microdeletion. Their frequencies were independent of maternal age. These data have implications for cell-free DNA screening tests design and justify prenatal screening for 22q11 deletion, as early recognition of DGS improves its prognosis.

Molecular analysis of products of conception obtained by hysteroembryoscopy from infertile couples.

PURPOSE: To analyze the molecular cytogenetic data obtained from products of conception (POC) obtained by selective biopsy of first trimester miscarriages and to estimate the rate of chromosomal anomalies in miscarriages from pregnancies achieved by natural conception (NC) or by assisted reproductive technology (ART) interventions. METHODS: We used KaryoLite™ BoBs™ (PerkinElmer LAS, Wallac, Turku, Finland) technology to analyze 189 samples from ART or NC pregnancies. RESULTS: All POC were successfully evaluated. A higher incidence of chromosomal abnormalities was observed in POC after ART using the patient's own oocytes than from NC pregnancies (62.7% vs. 40.6%; p < 0.05). The lowest incidence of chromosomal abnormalities was observed in POCs ART using donor eggs from women younger than 35 years (12.8%). No statistical differences in the percentage of abnormal miscarriages were observed in correlation with sperm concentration: a sperm concentration less than 5 million/mL produced 75% abnormal results and a concentration higher than 5 million/mL produced 51%. CONCLUSIONS: POC analysis is essential to determine the cause of pregnancy loss. Using culture-independent molecular biology techniques to analyze POCs avoids limitations such as growth failure and reduces the time required for analysis. Selective biopsy of fetal tissue by hysteroembryoscopy avoids the risk of misdiagnosis due to maternal cell contamination. Our results show that maternal age, sperm quality, and ART-assisted pregnancies are risk factors for abnormal gestations.

New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization.

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5-54.2%) and pregnancy rates per transfer (range: 46.0-62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.

BACs-on-Beads technology: a reliable test for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.

Assessment of sperm using mRNA microarray technology.

The aim of this work is to review the need for new diagnostic tools for sperm, the relevance of sperm mRNA in reproductive success, and the potential of a state-of-the-art microarray-based diagnostic tool for improving reproductive results.

Differential transcriptomic profile in spermatozoa achieving pregnancy or not via ICSI.

Basic sperm analysis is limited as a method of estimating pregnancy. This study's objective was use of microarray technology to differentiate the gene expressions of spermatozoa that achieved pregnancy in an intracytoplasmic sperm injection (ICSI)cycle in an oocyte donation programme with those that did not achieve pregnancy. A study of nested cases and controls was designed to evaluate fresh and frozen spermatozoa from infertile males undergoing ICSI with donor oocytes. The global genome expression of pooled samples from each group (achieving pregnancy versus those that didn't, from fresh or frozen spermatozoa)was compared using microarray analysis. The level of expression of some of the transcripts from fresh spermatozoa was shown to differ for those that achieved pregnancy versus those that didn't. Additionally, exclusively expressed transcripts were identified for both outcome groups. Analysis of frozen spermatozoa didn't reveal differential expression, but exclusively expressed transcripts were detected. Lists of the transcripts were systematically analysed using different databases in order to provide information about them and their relationship with male fertility. The results revealed profound differences between the expression profiles of spermatozoa that resulted in pregnancy versus those that didn't. These differences may explain ICSI failure associated with male factor infertility.

Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality.

OBJECTIVE: To quantify the effect of sperm DNA fragmentation (SDF) on reproductive outcome by evaluating the most statistically significant bias factors using logistic regression. DESIGN: Prospective blind observational cohort study. SETTING: University affiliated private IVF unit. PATIENT(S): Two hundred ten male partners of couples undergoing in vitro fertilization (IVF) or first intracytoplasmic sperm injection (ICSI) cycles with fresh or thawed sperm with the women's own or donated oocytes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): SDF determined before and after swim-up (n=420), odds ratio calculated of the effect of an increase of one unit of SDF on pregnancy, and stratified regression analysis performed to evaluate the confusion effect of oocyte quality, sperm origin, and the fertilization procedure. RESULT(S): The effect of SDF on pregnancy was not affected by sperm origin (fresh or thawed) or fertilization procedure when measured both before and after swim-up. When oocytes from infertile patients were employed, SDF had a statistically significant negative impact on chance of pregnancy. For every 10% increase in SDF, the probability of not achieving pregnancy increased by 1.31. When donated oocytes were employed, SDF did not have a statistically significant effect. CONCLUSION(S): The effect of SDF on the probability of pregnancy can be calculated independent of the fertilization procedure or sperm origin. Oocyte quality conditions the extent of the negative impact of SDF on pregnancy; this can be overcome when good quality oocytes are employed.

Ontological evaluation of transcriptional differences between sperm of infertile males and fertile donors using microarray analysis.

PURPOSE: To catalogue Gene Ontology terms in the sperm of infertile human males vs. donors of proven fertility by analyzing five samples from each of the two groups (five aliquots from fresh sperm and five post-swim-up). METHODS: Microarray technology was employed to study the mRNA profile of both fresh and post-swim-up pooled samples from infertile males and donors. RESULTS: Genes that were differentially expressed in the two populations and expressed in only one of two were analyzed to determine the gene products in terms of their associated Gene Ontology terms. Each group presented a different number and pattern of Gene Ontology terms. CONCLUSIONS: We found differences in Gene Ontology terms between the two groups. These differences could potentially be employed to establish markers of fertility success and to identify cellular processes and complex systems related with male infertility.

The transcriptome of spermatozoa used in homologous intrauterine insemination varies considerably between samples that achieve pregnancy and those that do not.

OBJECTIVE: To differentiate transcripts' expression in the sperm from patients who achieved pregnancy in their first IUI cycle from those who did not. Basic sperm analysis is limited to forecasting pregnancies by means of assisted reproduction. New assays, such as microarray analysis, are potential predictive tools for this purpose. DESIGN: Nested case-control study. SETTING: University-affiliated private setting. PATIENT(S): Twenty sperm samples were obtained from infertile males undergoing their first IUI cycle with healthy partners. Sperm samples with which pregnancy was achieved (P; n=10) and those with which it was not achieved (NP; n=10) were identified and their respective messenger RNA expression profiles were compared. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using microarrays, global genome expression was compared in pooled samples from each group. Results were evaluated to detect differentially expressed transcripts (TDEs; FC>2; P<0.05) and to identify those transcripts that were expressed in only one of the groups (exclusive transcripts [ETs]). RESULT(S): In group P, 756 TDEs presented increased expression, whereas 194 in group NP were found to be overexpressed. Furthermore, we found 741 ETs that were expressed only in group P and 976 that were expressed only in group NP. CONCLUSION(S): Results reveal profound differences between expression profiles of sperm samples that impregnate successfully and those that do not. These differences might improve the predictive power of sperm evaluation to estimate IUI success by complementing the basic sperm analysis.

Relevance of testicular sperm DNA oxidation for the outcome of ovum donation cycles.

OBJECTIVE: To determine the relevance of sperm DNA oxidation caused by free radicals in samples obtained via testicular biopsies by means of flow cytometry by correlating the measurements of 8-hydroxy-2'-deoxyguanosine (8-OHdG) with embryo features and pregnancy achievement. DESIGN: Prospective cross-sectional study. SETTING: Private University-affiliated setting. PATIENT(S): Fifty-seven azoospermic patients undergoing testicular sperm extraction (TESE) were analyzed in their corresponding assisted reporductive technology cycles using ovum donation to standardize female's characteristics. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantification of the adduct 8-OHdG in testicular tissue samples, and its effect on markers of embryo quality and reproductive success, and its relevance as marker of TESE sperm quality. RESULT(S): We found the status of sperm DNA oxidation to have very little clinical relevance for several parameters of embryo quality, fertilization rates, early (days 2-3) and late (days 5-6) development, and achievement of pregnancy. CONCLUSION(S): The TESE obtained cells from azoospermic males do not possess a DNA oxidation status of significant importance in the success of assisted reproduction treatments, as determined by 8-OHdG measurement of each category of cell ploidy.

Contribution of sperm molecular features to embryo quality and assisted reproduction success.

Semen analysis, as stated by the World Health Organization, is the only accepted tool to assess male fertility. However its predictive value to assess male capacity to initiate a pregnancy is limited. With the introduction of IVF (especially via intracytoplasmic sperm injection), infertility caused by diminished sperm production is frequently solved, but knowledge of sperm physiology remains very poor. Moreover, a percentage of males with apparently normal semen are unable to impregnate healthy women. Therefore, improvements in the diagnostic tools to assess male fertility potential are necessary. The aim of this review is to describe sperm molecular factors implicated in male fertility, demonstrated by their role in sperm physiology, the molecular differences found between fertile and infertile males, or by their influence on the results obtained in assisted reproduction treatments in terms of embryo quality and pregnancy achievement. From a search and objective evaluation of the currently available evidence, it is concluded that there is no unique factor able to predict male fertility, but several molecular factors are involved in sperm function and can potentially be considered as fertility markers. In this context, a complex molecular tool designed to analyse a battery of parameters seems to be necessary.