Semen quality of young adult ICSI offspring: the first results.

STUDY QUESTION: What is the semen quality of young adult men who were conceived 18-22 years ago by ICSI for male infertility? SUMMARY ANSWER: In this cohort of 54 young adult ICSI men, median sperm concentration, total sperm count and total motile sperm count were significantly lower than in spontaneously conceived peers. WHAT IS KNOWN ALREADY: The oldest ICSI offspring cohort worldwide has recently reached adulthood. Hence, their reproductive health can now be investigated. Since these children were conceived by ICSI because of severe male-factor infertility, there is reasonable concern that male offspring have inherited the deficient spermatogenesis from their fathers. Previously normal pubertal development and adequate Sertoli and Leydig cell function have been described in pubertal ICSI boys; however, no information on their sperm quality is currently available. STUDY DESIGN, SIZE, DURATION: This study was conducted at UZ Brussel between March 2013 and April 2016 and is part of a large follow-up project focussing on reproductive and metabolic health of young adults, between 18 and 22 years and conceived after ICSI with ejaculated sperm. Results of both a physical examination and semen analysis were compared between young ICSI men being part of a longitudinally followed cohort and spontaneously conceived controls who were recruited cross-sectionally. PARTICIPANTS/MATERIALS, SETTING, METHOD: Results of a single semen sample in 54 young adult ICSI men and 57 spontaneously conceived men are reported. All young adults were individually assessed, and the results of their physical examination were completed by questionnaires. Data were analysed by multiple linear and logistic regression, adjusted for covariates. In addition, semen parameters of the ICSI fathers dating back from their ICSI treatment application were analysed for correlations. MAIN RESULTS AND THE ROLE OF CHANCE: Young ICSI adults had a lower median sperm concentration (17.7 million/ml), lower median total sperm count (31.9 million) and lower median total motile sperm count (12.7 million) in comparison to spontaneously conceived peers (37.0 million/ml; 86.8 million; 38.6 million, respectively). The median percentage progressive and total motility, median percentage normal morphology and median semen volume were not significantly different between these groups. After adjustment for confounders (age, BMI, genital malformations, time from ejaculation to analysis, abstinence period), the statistically significant differences between ICSI men and spontaneously conceived peers remained: an almost doubled sperm concentration in spontaneously conceived peers in comparison to ICSI men (ratio 1.9, 95% CI 1.1-3.2) and a two-fold lower total sperm count (ratio 2.3, 95% CI 1.3-4.1) and total motile count (ratio 2.1, 95% CI 1.2-3.6) in ICSI men compared to controls were found. Furthermore, compared to men born after spontaneous conception, ICSI men were nearly three times more likely to have sperm concentrations below the WHO reference value of 15 million/ml (adjusted odds ratio (AOR) 2.7; 95% CI 1.1-6.7) and four times more likely to have total sperm counts below 39 million (AOR 4.3; 95% CI 1.7-11.3). In this small group of 54 father-son pairs, a weak negative correlation between total sperm count in fathers and their sons was found. LIMITATIONS, REASONS FOR CAUTION: The main limitation is the small study population. Also, the results of this study where ICSI was performed with ejaculated sperm and for male-factor infertility cannot be generalized to all ICSI offspring because the indications for ICSI have nowadays been extended and ICSI is also being performed with non-ejaculated sperm and reported differences may thus either decrease or increase. WIDER IMPLICATIONS OF THE FINDINGS: These first results in a small group of ICSI men indicate a lower semen quantity and quality in young adults born after ICSI for male infertility in their fathers. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts, all issued by the Vrije Universiteit Brussel (VUB). All co-authors except M.B. and H.T. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough and Merck for establishing the database for follow-up research and organizing the data collection. The institution of H.T. has received research grants from the Research Fund of Flanders (FWO), an unconditional grant from Ferring for research on testicular stem cells and research grants from Ferring, Merck, MSD, Roche, Besins, Goodlife and Cook for several research projects in female infertility. H.T. has received consultancy fees from Finox, Abbott and ObsEva for research projects in female infertility.

[Genetics and male infertility]. / Genetica van de mannelijke onvruchtbaarheid.

Infertility is a problem affecting many couples with a child wish. In about half of these couples a male factor is (co-) responsible for the fertility concern. For part of these patients a genetic factor will be the underlying cause of the problems. This paper gives an overview of the studies performed in the Department of Embryology and Genetics of the Vrije Universiteit Brussel and the Centre for Medical Genetics of UZ Brussel in order to gain more insight into the genetic causes of male infertility. The studies, focusing on men with fertility problems, can be subdivided into three groups: studies on deletions on the long arm of the Y chromosome, studies on X-linked genes and studies on autosomal genes. It is obvious that Yq microdeletions should be considered as a cause of male infertility. Only for patients with a complete AZFc deletion, a small number of spermatozoa can be retrieved. However, even for these patients assisted reproductive technologies are necessary. Complete AZF deletions are found in 4.6% of the patients visiting the centres for Reproductive Medicine and Medical Genetics of the UZ Brussel and for whom no other cause of the fertility problems have been detected. Taken into consideration this low prevalence of Yq microdeletions, it is obvious that also other factors, including genetic factors, must be causing fertility problems. Potentially, gr/gr deletions (partial deletions of the AZFc region) might influence the fertility status of the patients. It remains, however, unclear which of the genes located in the deleted regions are important for the progression of spermatogenesis, in case of partial or complete AZF deletions. In our studies we have also investigated mutations in genes located on the X chromosome. In analogy to the Y chromosome, the X chromosome is interesting in view of studying male infertility since men only have a single copy of the sex chromosomes. As a consequence, mutations in genes crucial for spermatogenesis will have an immediate impact on the sperm production. The genes NXF2, USP26 and TAF7L were investigated for the presence of mutations. All observed single nucleotide changes were also present in control samples, questioning their relationship with male infertility. We also studied five autosomal genes: SYCP3, MSH4, DNMT3L, STRA8 and ETV5. Only for the genes STRA8 and ETV5, changes were detected that were absent in a control population existing of men with normozoospermia. Functional analysis of the changes in ETV5 and the localization of the change observed in STRA8 showed that also these alterations were probably not the cause of the fertility problems in these men. It can be concluded that mutations are rarely detected in men with fertility problems. This low frequency of mutations has also been confirmed in several published studies. Therefore, further research is necessary to determine the impact of genetic causes on male infertility.

CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Assessment of radioimmunoassay of serum creatine kinase BB (CK-BB) as a tumor marker: studies in patients with various cancers and a comparison of CK-BB concentrations to prostate acid phosphatase concentrations.

The association between cancer and the BB isoenzyme of creatine kinase (CK-BB) was investigated, and the possibility of the role of CK-BB as a tumor marker was assessed. With the use of a specific radioimmunoassay, the concentration of CK-BB was measured in 524 sera (obtained from the National Cancer Institute-Mayo Clinic Serum Diagnostic Bank) from patients with a variety of benign and malignant disorders. In 79 of these sera, the results of radioimmunoassay for CK-BB were compared to those of three radioimmunoassays for prostate acid phosphatase. Abnormal CK-BB concentrations occurred in only about 11% of the 366 cancer patients. Some groups of cancer patients had higher rates; e.g., the CK-BB concentration was elevated in 29% of the prostate cancer patients. However, prostate acid phosphatase was abnormal in 65% of the patients with prostate carcinoma--a considerable higher fraction than that found with CK-BB. Findings in patients with benign and malignant gastrointestinal diseases indicate that CK-BB complements carcinoembryonic antigen data and might be useful as part of a tumor marker panel.

Creatine kinase isozymes in the serum and CSF of schizophrenic patients.

Newly admitted psychotic patients often have elevations of serum creatine kinase (CK) enzymatic activity. Previous studies indicate that this increase consists of the muscle (MM) isozyme, and increases in the brain (BB) isozyme have not been observed. Using sensitive and specific radioimmunoassays that detect both active and inactive enzyme, we measured CK-MM and CK-BB in the serum and CSF of 100 patients with schizophrenia who were not newly admitted but whose conditions varied from acute to chronic to determine whether CK-MM or CK-BB appears in the CSF and whether CK-BB can be found in the serum of these patients. We found no unusual concentrations of either isozyme in CSF. We did observe a few elevations in serum CK-BB levels, but this test does not appear to be of diagnostic value for schizophrenic patients who are not newly admitted.

Ectopic adrenocorticotropin production: disappearance after removal of inflammatory tissue.

A patient presented with the clinical and laboratory features of the ectopic ACTH syndrome. No ACTH-producing tumor was found, and bilateral adrenalectomy was performed to correct the hypercortisolism. Six months later, the removal of a pseudotumor containing only fat and inflammatory tissue resulted in normalization of both basal plasma ACTH levels and the ACTH feedback response to cortisol infusion. It is suggested that the leukocytes in the inflammatory tissue were the source of the ectopic ACTH production.

Regulation of maternal thyroid during pregnancy.

A prospective study was undertaken in 606 healthy women during pregnancy to evaluate the changes occurring in maternal thyroid economy as a result of 1) the increased thyroid hormone-binding capacity of serum, 2) the effects of increased levels of hCG on TSH and on the thyroid, and 3) a marginally low iodine intake in the population (50-75 micrograms/day). Four main features were observed. First, thyroidal activity adjusted to the marked increase in serum T4-binding globulin: pregnancy was accompanied by an overall reduction in the T4/T4-binding globulin ratio, with lower free T4 and T3 levels, although in most cases free hormone levels remained within the normal range. The adjustment of thyroidal output of T4 and T3 did not occur similarly in all subjects. In approximately one third of the women, there was relative hypothyroxinemia, higher T3/T4 ratios (presumably indicating preferential T3 secretion), and higher, although normal, serum TSH concentrations. Second, high hCG levels were associated with thyroid stimulation, both functionally (lower serum TSH) and anatomically (increased thyroid size). The data are consistent with a TSH-like effect of hCG on the thyroid. Hence, regulation of the maternal thyroid is complex, resulting from both elevated hCG (mainly in the first half of gestation) and increasing TSH (mainly in the second half of gestation). Third, a significant increase in serum thyroglobulin levels was observed throughout gestation, especially during the last trimester. Fourth, increased thyroid volume was common, and goiter formation not uncommon (goiter was found in 9% of women at delivery). In conclusion, the alterations in maternal thyroid function during gestation are intricate and far from fully understood. In areas of marginally low iodine intake, gestation is associated in a significant number of women with relative hypothyroxinemia, increased thyroglobulin, and enlarged thyroid.

Protein-energy malnutrition and anemia in Kivu.

Protein-energy malnutrition in Kivu is associated with a discrete normocytic, normochromic anemia. An attempt to define the physiopathology of this anemia disclosed the following results. As compared with local controls, both iron and total iron binding capacity were low, but with siderophilin saturation and sideroblast counts either normal or elevated; serum and erythrocyte folate was normal, plasma vitamin B12 was normal or elevated, and serum ascorbic acid was normal or elevated. The riboflavin nutritional status was normal. During refeeding, iron and riboflavin deficiencies became apparent. Characteristic findings on admission were the presence of giant erythroblasts and a diminished erythrocyte survival time implicated to an intracorpuscular hemolysis. Two results from the present study could contribute to explanation for the aforementioned abnormalities: low plasma vitamin E levels and, perhaps more importantly, low plasma selenium levels. In conclusion, the anemia of protein-energy malnutrition, as observed in Kivu, is a classifiable nonadaptive anemia that cannot be explained by isolated iron or vitamin deficiencies and whose physiopathology is distinct from that of the anemia of chronic disorders. It is suggested that a selenium deficiency may play an important role in the pathogenesis of this anemia.

Embryo freezing after intracytoplasmic sperm injection.

This study reports on the safety and efficiency of the cryopreservation of human embryos obtained after intracytoplasmic sperm injection. For this, we evaluated the morphological survival, the capacity of the surviving embryo to develop further in vitro and in vivo. After freezing-thawing embryos obtained after ICSI, 40% of the embryos do not survive the cryopreservation procedure. After selective transfer of further cleaving frozen-thawed embryos, pregnancy loss was 31% (subclinical pregnancy rate of 13% and miscarriage rate of 18%). As a result the livebirth rate per transferred embryos and per thawed embryo was 7 and 3% respectively. Obstetric outcome as well as further follow-up of the children born indicate that cryopreservation of ICSI embryos is a safe procedure, long term follow-up of the children born however is still warranted.

PGD in the lab for triplet repeat diseases - myotonic dystrophy, Huntington's disease and Fragile-X syndrome.

Myotonic dystrophy (DM), Huntington's disease (HD) and Fragile X syndrome (FRAXA) are three monogenic disease which are caused by so-called dynamic mutations. These mutations are caused by triplet repeats inside or in the vicinity of the gene which have the tendency to expand beyond the normal range thus disrupting the normal functioning of the gene. We describe here our experiences from 1995 to May 2000 with PGD for these three triplet repeat diseases.

The effect of pentoxifylline on in-vitro fertilization in the presence of anti-sperm antibodies.

In cases of severe immunological male-factor infertility, impairment of spermatozoal motility and of acrosome reaction resulting in reduced fertilization capacity have been described by several authors. The present study investigated the use of pentoxifylline in enhancing in-vitro fertilization (IVF) in the presence of anti-sperm antibodies. Thirty-seven IVF cycles were conducted in 28 different couples suffering from immunological male-factor infertility with at least 50% antibody-coated spermatozoa. Sibling oocytes were inseminated at random with spermatozoa incubated with or without 3.6 mM pentoxifylline after selection by a Percoll gradient. No difference in motility of the final sperm preparations was observed prior to insemination. Fertilization rate, cleavage rate and embryo quality were similar in both treatment and control groups. Nine out of ten pregnancies were achieved after the replacement of embryos both from the treatment and control group. Although pentoxifylline is known to enhance motility in-vitro and to promote induced acrosomal loss, its indiscriminate use failed to improve IVF performance in patients with anti-sperm antibodies. Further research may be necessary in order to elucidate whether a given subpopulation of these patients may benefit from a selective application of pentoxifylline.

Successful in vitro fertilization with sperm cells from a man with immune infertility.

In vitro fertilization and embryo transfer was carried out in a couple that has been infertile for 7 years. High titers of antisperm autoantibodies in the husband's serum are (table, see text) believed to be the main cause of their infertility. Prolonged methylprednisolone treatment of the man combined with artificial insemination remained unsuccessful. In two IVF attempts embryos were obtained and replaced using capacitated sperm of the husband. A pregnancy was established in the second IVF cycle, but a spontaneous abortion followed after 6 weeks of amenorrhea.

Survey on cryopreservation.

In this survey on cryopreservation of human embryos and oocytes, the results from 24 groups that reported replacements of cryopreserved embryos by the end of 1986 were summarized. So far 163 pregnancies have been reported, 65 children have been born, and 61 pregnancies were ongoing at the time of tabulation. The real impact of freezing and thawing of human embryos and oocytes in the treatment of infertility will require a careful analysis of all factors influencing the cryopreservation.

In vitro fertilization in couples with previous fertilization failure using sperm incubated with pentoxifylline and 2-deoxyadenosine.

OBJECTIVE: To evaluate whether incubation of spermatozoa with both pentoxifylline and 2-deoxyadenosine would improve fertilization rates in couples with previous IVF failure. DESIGN: Autocontrolled design in which sibling oocytes were inseminated at random in vitro with spermatozoa treated or not treated by pentoxifylline and 2-deoxyadenosine. MEAN OUTCOME MEASURES: Oocyte quality, sperm motility, fertilization in vitro, and embryo quality. RESULTS: Sperm motility was found optimized by metabolic stimulation using pentoxifylline and 2-deoxyadenosine. The mean fertilization rate per patient was 33.1% in the treatment group compared with 37.0% in the control group. The mean cleavage rate per patient was 79.6% for treatment versus 68.7% for control embryos. No differences in embryo quality were noted. CONCLUSION: The results of this study demonstrate that an indiscriminate use of pentoxifylline and 2-deoxyadenosine is not beneficial to fertilization in couples with previous IVF failure. Further prospective research may be needed to assess the benefit of pentoxifylline and 2-deoxyadenosine in patients selected by preliminary functional in vitro tests.

Amplification of exon 11 of the gene for the alpha-chain of beta-N-acetylhexosaminidase in single human blastomeres.

OBJECTIVE: To evaluate the possibility of using polymerase chain reaction (PCR) technology for preimplantation diagnosis for Tay-Sachs disease. DESIGN: Polymerase chain reaction on single human blastomeres. SETTING: Bad quality embryos from IVF analyzed in an academic research genetics lab. INTERVENTIONS: Patients underwent standard IVF procedures as infertility treatment. RESULTS: Amplification was seen in 89.5% of the blastomeres; only one blank was contaminated. CONCLUSION: The technique of PCR on single blastomeres is ready to be used in clinical preimplantation diagnosis for Tay-Sachs disease.

Human serum albumin versus serum: a comparative study on embryo transfer medium.

OBJECTIVE: This study has examined the efficacy of human albumin as a replacement for serum in embryo transfer (ET) medium. DESIGN: In a prospective, randomized study, 224 cycles were analyzed. Patients were randomly divided into three groups: in group A, embryos were transferred in 75% serum; in group B, in 8% serum; and in group C, 2.25% human serum albumin (HSA). SETTING: All the ETs were performed in patients from the in vitro fertilization program of the Center for Reproductive Medicine, Vrije Universiteit Brussel, Belgium. PATIENTS, PARTICIPANTS: In 194 patients, 224 ETs were performed. The patients were selected when they had at least one good-quality embryo. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Use of albumin in culture medium resulted in higher pregnancy rates (PRs). To further simplify the procedure, albumin was also used in ET medium. RESULTS: An overall PR of 43% in group A, 27% in group B, and 36% in group C was achieved. No significant difference was observed in PRs between three different transfer media. CONCLUSIONS: We conclude that HSA is a safe and suitable replacement for serum, both in embryo culture and in the transfer medium.

The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes.

OBJECTIVE: To assess the effect of propofol on fertilization and early embryo development in a mouse IVF model. DESIGN: Controlled study. SETTING: Mouse IVF. INTERVENTION(S): Mouse oocytes were exposed in vitro to propofol at a concentration of 0 (control), 50, 250, 500, 1,000, or 5,000 ng/mL for 30 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the blastocyst stage was monitored daily. The potential to activate parthenogenetically oocytes also was evaluated by looking for spontaneous extrusion of the second polar body or development to the two-cell stage. In a second step, a pure propofol solution was added to culture medium and used as a standard. MAIN OUTCOME MEASURE(S): Two-cell and blastocyst-stage embryo. RESULT(S): Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of propofol solution in the medium, (i.e., 3% to 41%) in comparison with the control group (76%). Exposure of unfertilized oocytes for 30 minutes to propofol results in a parthenogenetic activation of 33% to 60%, which was significantly higher than the control (10%). When oocytes were kept in propofol for 24 hours, a mean of 30% of activation was observed as compared with 0.5% for the control. CONCLUSION(S): We can conclude from these experiments that even a brief exposure of cumulus-enclosed oocytes to a low concentration of propofol is deleterious to subsequent cleavage. Exposure of unfertilized oocytes to propofol results in a high degree of parthenogenetic activation.

Treatment of infertility because of oligoasthenoteratospermia by transcervical intrauterine insemination of motile spermatozoa.

Fifty-three couples with longstanding infertility, the woman being potentially fertile and the man having poor-quality semen, were admitted to a therapeutic trial. A high intrauterine instillation of a washed motile fraction of spermatozoa from the subfertile man was performed in the woman on the day of ovulation. After 130 treatment cycles, 9 of the 53 women had conceived, all within 3 cycles. This simple and painless procedure can be considered an alternative treatment in oligoasthenoteratospermia.

Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes.

OBJECTIVE: To analyze embryos after in vitro maturation by investigating their nuclear status and cytogenetic constitution. DESIGN: Prospective randomized laboratory study. SETTING: Reproductive medicine unit in an academic hospital. PATIENT(S): Patients with male and tubal factor infertility undergoing fertility treatment. INTERVENTION(S): Denuded immature oocytes (n = 75) were matured in vitro for 24-30 hours, and intracytoplasmic sperm injection was performed 30 hours after oocyte retrieval. Fluorescence in situ hybridization was performed on the produced embryos. MAIN OUTCOME MEASURE(S): Blastomere content of the total embryo. RESULT(S): The in vitro-matured oocytes showed a similar fertilization rate as the in vivo-matured oocytes, but with a higher incidence of noncleavage (21.0%). In addition, 26.7% of these embryos arrested at the first mitotic division. Thirty embryos were processed for fluorescence in situ hybridization; only 6.7% had all mononuclear blastomeres, 30.0% had at least one binuclear blastomere, 43.3% had at least one multinuclear blastomere, and 56.6% contained anuclear cells. The chromosomal constitution was analyzed in 14 embryos, and chromosomal anomalies were found in 11 (78.5%). CONCLUSION(S): Germinal vesicle oocytes retrieved from superovulated patients and cultured in vitro for a short time had the ability to resume meiosis and achieve fertilization. However, arrest of embryo development was common. These embryos showed a high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities.