Genome and Epigenome Disorders and Male Infertility: Feedback from 15 Years of Clinical and Research Experience.

Infertility affects around 20% of couples of reproductive age; however, in some societies, as many as one-third of couples are unable to conceive. Different factors contribute to the decline of male fertility, such us environmental and professional exposure to endocrine disruptors, oxidative stress, and life habits with the risk of de novo epigenetics dysregulation. Since the fantastic development of new "omes and omics" technologies, the contribution of inherited or de novo genomes and epigenome disorders to male infertility have been further elucidated. Many other techniques have become available to andrology laboratories for the investigation of genome and epigenome integrity and the maturation and the competency of spermatozoa. All these new methods of assessment are highlighting the importance of genetics and epigenetics investigation for assisted reproduction pathology and for supporting professionals in counselling patients and proposing different management strategies for male infertility. This aims to improve clinical outcomes while minimizing the risk of genetics or health problems at birth.

How to overcome male infertility after 40: Influence of paternal age on fertility.

The recent trend toward delayed parenthood raises major safety concerns because of the adverse effects of aging on couple fertility. Studies have demonstrated that aging clearly affects female fertility, but can also affect male fertility. Although several theories have been proposed, the exact mechanisms responsible for the observed age-related decline in male fertility remain to be elucidated. It has been shown that advanced paternal age (PA) is associated with reduced semen volume as well as, reduced sperm count, motility and morphology. Recent studies have also reported that paternal aging is associated with a significant increase in the prevalence of both genomic and epigenomic sperm defects. In the context of natural and intrauterine insemination (IUI) conception, advanced paternal age has been associated with lower pregnancy rates and increased rates of spontaneous abortion (independent of maternal age). In IVF and oocyte donation programs, a significant decrease in late blastocyst development has been seen in those cycles using spermatozoa of men older than 55. However, no significant relationship between paternal age and IVF or ICSI pregnancy rates has been observed. Although there are no treatments that can fully restore the age-related decline in male fertility, various measures have been shown to optimize male fertility potential. Specific therapies (e.g. varicocelectomy) and lifestyle changes (e.g. dietary antioxidant supplements) may help minimize some of the age-related deleterious effects on spermatogenesis, such as, oxidative stress and endocrine abnormalities.

Management of infertility in women over 40.

Women's fertility potential is declining with age because of multiples intrinsic and extrinsic factors such as life style, oxidative stress and/or endocrine disruptors and is affecting the ability of these women to conceive naturally. This declining fertility potential and the late age of motherhood is increasing significantly the number of patients consulting infertility specialists. Different strategies of investigation and management are proposed to patients over 40 in order to overcome their infertility and improve the live birth rate in these patients. Intra Uterine Insemination (IUI) in women over 40 is associated with a low rate of ongoing pregnancy and IUI should not therefore be offered always as the first line of treatment. When the predictive factors are positive IVF/ICSI seem to be good alternatives until 43 years of age. Customized ovarian stimulation and flexible laboratory methods such as in vitro maturation (IVM), preimplantation genetic diagnosis (PGD), embryo vitrification and transfer after thawing in subsequent natural or artificial cycles can improve the success rate of ART in patients over 40. Meanwhile, oocyte and embryos donation remain good options for patient over 40 with a bad prognosis and can lead to successful ongoing pregnancies until 45 years of age. Ovarian tissue cryopreservation, oocyte vitrification at the germinal vesicle (GV) stage or metaphase II stage present a breakthrough for fertility preservation but the ideal age for starting fertility preservation is still debated as well as the minimum number of oocytes to be vitrified in order to optimize the chances of pregnancy when needed at an older age. This manuscript reports the results of our own experience from patients older than 40 in the light of the published data and discusses the different therapeutic alternatives which can be proposed to patients over 40 consulting ART centres.

Low birth defects by deselecting abnormal spermatozoa before ICSI.

Consistent evidence from meta-analysis has linked assisted conception by IVF, and particularly intracytoplasmic sperm injection (ICSI), with an increased risk of major birth defects. To compare the risk of major malformations of children born after standard ICSI and after intracytoplasmic injection of morphologically selected spermatozoa (IMSI), a prospective population-based study was conducted from 2005 to 2010. ICSI and IMSI were performed in only one assisted reproduction unit according to its classification of spermatozoa and using fresh semen. Medical data and follow up during 2 years of 1028 infants were collected. Major malformations were identified and classified by an external independent physician. The two groups were similar concerning the parents' age, treatment, number of oocytes recovered, days of transfer, gestational age and birthweight. However, major malformations were significantly lower with IMSI (6/450, 1.33%) versus ICSI (22/578, 3.80%; adjusted odds ratio 0.35, 95% confidence interval 0.14-0.87, P=0.014), mainly affecting boys (adjusted odds ratio 2.84, 95% confidence interval 1.24-6.53, P=0.009). In conclusion, the significantly decreased risk of major birth defects associated with IMSI remained decreased after multivariate adjustment and highlights the beneficial effect of sperm selection before ICSI.

Advantages of the two-step embryo transfer strategy in human IVF/ICSI cycles.

The aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10(-3)). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32-3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.

Culture systems: embryo co-culture.

During the 1970s, domestic animal biotechnology, i.e., embryo transfer in farm animals, was confronted with the problem of embryonic developmental arrest observed in vitro, especially during the cycle in which maternal to zygotic transition (MZT) cycle takes place. In farm animals, obtaining blastocysts is mandatory, as transfer at earlier stages results in expulsion of the embryo from the vagina. In humans, the first attempts to obtain blastocysts with classical culture media were disappointing, and the use of a coculture strategy was naturally tempting: the first significant results of successful blastocyst development were obtained in the early 1980s, using trophoblastic tissue as a feeder layer in order to mimic an autocrine embryotrophic system. The next supporting cell systems were based on oviduct epithelial cells and uterine cells in order to achieve a paracrine effect. Non-hormone dependence was then demonstrated with the use of prepubertal cells, and finally with the use of established cell lines of nongenital origin (African Green Monkey Kidney, Vero cells). The embryotrophic properties are linked to features of "transport epithelia." Vero cells have been extensively used in human ART, and most of our knowledge about the human blastocyst was gathered with the use of this technology. Coculture is still in current use, but with systems that employ autologous uterine cells. Results following the use of this technology in human ART are superior to those observed with the use of sequential media. The benefit is linked to the release of free radical scavengers and growth factors by the feeder cells. In animal biotechnology, an important part of the "precious embryos," i.e., those resulting from cloning technology, involves coculture with buffalo rat liver (BRL) cells or Vero cells.

Correlation between DNA defect and sperm-head morphology.

The utility of sperm DNA testing remains controversial. However, it may be helpful in couples with unexplained failures of multiple assisted reproductive techniques and/or recurrent abortions. This study analysed 10,400 spermatozoa of 26 patients for sperm-head morphology with high-magnification microscopy, DNA fragmentation and sperm chromatin decondensation. A significant negative correlation was demonstrated between sperm-parameters and abnormal sperm-head morphology as assessed by high magnification (score 0 according to this study's classification): concentration (r=-0.41; P=0.03), motility (r=-0.42; P=0.03), morphology (r=-0.63; P=0.0008). No correlation was found with DNA fragmentation. However, the sperm chromatin-decondensation rate of score-0 spermatozoa was twice as high as the controls (19.5% versus 10.1%; P<0.0001). This observation suggests that score-0 spermatozoa should not be selected for intracytoplasmic sperm injection.

Impact of freezing/thawing technique on sperm DNA integrity in HIV-1 patients.

INTRODUCTION: According french legislation, sperm freezing/thawing procedures are used to prevent ART contaminations in couple with HIV-1 infected men. We determined sperm nuclear fragmentation rate before and after selection and freezing/thawing in HIV-1 14 patients. METHODS: Two groups of patients were studied: 20 control patients with normal sperm (group 1) and without viral infection and 20 fertile treated HIV-1 patients (group 2). DNA fragmentation was evaluated using terminal uridine nick end labeling, before and after gradient selection, and after cryopreservation and thawing procedures. RESULTS: DNA fragmentation rates in fresh semen were increased in HIV patients (6.38% vs 3.39%) (p < 0.05) compared with control patients. After sperm migration, fragmentation rates were significantly lower (p < 0.0001) in the two groups compared with fresh sperm rates. After freezing/thawing, values were similar to those of fresh semen with an increased rate (p < 0.01) for HIV-1 patients, with respectively 3.40% and 5.18% rates in control and infected patients. HIV-1-infected patients treated by antiretroviral therapy showed a significant increase in sperm DNA fragmentation in fresh sperm and also after freezing/thawing procedures, but these two fragmentation rates were not significantly different. CONCLUSION: So, freezing/thawing procedures do not seem to impair sperm DNA and preserve probability of conception for couples with HIV-1 infected men.

Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination.

More than 17,000 intrauterine insemination (lUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.

Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (-19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

Beneficial effect of luteal-phase GnRH agonist administration on embryo implantation after ICSI in both GnRH agonist- and antagonist-treated ovarian stimulation cycles.

BACKGROUND: GnRH agonist was recently suggested as a novel luteal-phase support that may act at different levels, including the pituitary gonadotrophs, the endometrium and the embryo itself. This prospective randomized study evaluates the effect of GnRH agonist administered in the luteal phase on ICSI outcomes in both GnRH agonist- and GnRH antagonist-treated ovarian stimulation protocols. METHODS: Six hundred women about to undergo ovarian stimulation for ICSI (300 using a long GnRH agonist protocol and 300 using a GnRH antagonist protocol) were enrolled in this study. Patients treated with each of these two protocols were randomly assigned to receive a single injection of GnRH agonist or placebo 6 days after ICSI. Implantation and live birth rates were the primary outcomes. RESULTS: Administration of 0.1 mg of GnRH agonist triptorelin on day 6 after ICSI led to a significant improvement of implantation and live birth rates after ICSI as compared with placebo. In GnRH antagonist-treated ovarian stimulation cycles, luteal-phase GnRH agonist also increased ongoing pregnancy rate. Moreover, luteal-phase GnRH agonist administration increased luteal-phase serum HCG, estradiol and progesterone concentrations in both ovarian stimulation regimens. CONCLUSIONS: Luteal-phase GnRH agonist administration enhances ICSI clinical outcomes after GnRH agonist- and GnRH antagonist-treated ovarian stimulation cycles, possibly by a combination of effects on the embryo and the corpus luteum.

High-magnification ICSI overcomes paternal effect resistant to conventional ICSI.

Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.

Pregnancy and delivery after in vitro maturation of naked ICSI-GV oocytes with GH and transfer of a frozen thawed blastocyst: case report.

PURPOSE: To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. METHOD: All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37 degrees C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. RESULTS: The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. CONCLUSION: In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.

Improvement of delivery and live birth rates after ICSI in women aged >40 years by ovarian co-stimulation with growth hormone.

BACKGROUND: Growth hormone (GH) is required for ovarian follicular development, and its administration during ovarian stimulation improves pregnancy rate in cow and sheep. Data on the use of exogenous GH in human assisted reproduction treatment are inconsistent. This prospective randomized study evaluates the usefulness of GH administration in women of >40 years undergoing ovarian stimulation for assisted reproduction treatment. METHODS: One hundred women of >40 years undergoing assisted reproduction treatment were randomized between a GH treatment group and a placebo group. Assisted reproduction treatment outcomes were evaluated. RESULTS: In patients of the GH treatment group, a similar number of oocytes, embryos and pregnancies was achieved as compared with the placebo group. However, the patients treated with GH suffered fewer pregnancy losses, resulting in higher delivery and live birth rates. These patients also showed higher peak serum estradiol concentration and higher concentrations of GH and estradiol in pre-ovulatory follicular fluid as compared with the placebo group. CONCLUSIONS: Administration of GH during ovarian stimulation alleviates age-related decrease in assisted reproduction treatment efficiency. This effect appears to be mainly due to an improvement of oocyte developmental potential, but GH action on the uterus cannot be excluded.

Serum antimüllerian hormone/müllerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol.

OBJECTIVE: To test the hypothesis that the concentration of early follicular phase serum antimullerian hormone (AMH) or mullerian-inhibiting substance (MIS) is a useful marker of ovarian response and assisted reproductive technology (ART) outcome. DESIGN: Retrospective analysis of day 3 serum samples drawn before treatment. SETTING: Private ART program. PATIENT(S): One hundred nine consecutive serum samples from women younger than 42 years of age who were undergoing ovulation induction for IVF. INTERVENTION(S): Follicular aspiration for IVF after ovarian stimulation with FSH in a down-regulated cycle using GnRH-a treatment. MAIN OUTCOME MEASURE(S): Correlations between day 3 serum AMH/MIS, E2, FSH, inhibin B levels, and IVF outcome (i.e., number of retrieved mature oocytes, number and quality of embryos obtained, ongoing clinical pregnancy rates). Multivariate regression analysis on categorical data was performed to describe a predictive model of clinical pregnancy outcome. RESULT(S): Mean serum AMH/MIS value for clinical pregnancy (n = 38) was 2.4 ng/mL, in comparison to 1.1 ng/mL for those who did not become pregnant (n = 71). No differences were noted in mean values for day 3 FSH, inhibin B, or E2 between groups. Multivariate regression analysis demonstrated that day 3 serum AMH/MIS had the greatest independent contribution in predicting pregnancy outcomes. CONCLUSION(S): These data demonstrate a strong association between day 3 serum AMH/MIS level and IVF outcome in women younger than 42 years of age. Higher AMH/MIS concentrations are associated with a greater number of mature oocytes, a greater number of embryos, and ultimately a higher clinical pregnancy rate. Furthermore, AMH/MIS may offer greater prognostic value than other currently available serum markers of ART outcome.

Enhancement of embryo developmental potential by a single administration of GnRH agonist at the time of implantation.

BACKGROUND: Several reports have shown that inadvertent administration of a GnRH agonist in the luteal phase does not compromise pregnancy. Moreover, some studies suggested that, unexpectedly, the embryo developmental potential is improved in these conditions. This prospective controlled study was designed to test this hypothesis. METHODS: In an oocyte donation programme, oocytes from each donor (n = 138) were shared by two recipients, one of whom was given a single dose of a GnRH agonist (0.1 mg triptorelin) 6 days after ICSI, and the other received placebo at the same time. RESULTS: Oocyte recipients treated with GnRH agonist 6 days after ICSI had higher implantation (36.9 versus 25.1%), twin pregnancy (16.7 versus 3.6%), twin delivery (13.8 versus 2.2%) and birth (31.1 versus 21.5%) rates and similar miscarriage and abortion rates as compared with the placebo group. CONCLUSIONS: GnRH agonist administration at the time of implantation enhances embryo developmental potential, probably by a direct effect on the embryo.

Luteinizing hormone affects uterine receptivity independently of ovarian function.

Previous studies have suggested that LH, in addition to its well-known effects on the ovary, may exert direct effects on the uterus. This study evaluated the effects of mid-cycle administration of human chorionic gonadotrophin (HCG), which signals through the LH receptor, on endometrial thickness and uterine receptivity in two groups of women lacking ovarian activity and receiving embryos from an oocyte donation programme. Patients in one group still had ovulatory cycles, but their ovarian function was suppressed by pituitary down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist in the embryo transfer cycle, resulting in low endogenous LH concentrations. Patients in the other group were menopausal women whose pituitary function was not down-regulated in the embryo transfer cycle and whose endogenous LH concentrations were thus high. Patients in each of the two groups were randomized into two subgroups. Patients in one subgroup were given 5000 IU of HCG 2 days before oocyte recovery in the corresponding donor. Patients in the other subgroup received placebo at the same time. Oocytes from each donor were randomly distributed between one patient from the HCG subgroup and one patient from the placebo subgroup in each patient group. Endometrial growth and secretory transformation were stimulated by sequential treatment with oestradiol valerate and progesterone. In women with low endogenous LH receiving placebo, endometrial thickness stopped increasing at the beginning of secretory transformation. Mid-cycle HCG administration resulted in a continuous increase in endometrial thickness through this period, improved the implantation rate after embryo transfer in these women (30.6 versus 20.7%) and augmented the number of multiple pregnancies. No similar stagnation of endometrial thickness and no effects of mid-cycle HCG administration on endometrial thickness, the implantation rate and the number of multiple pregnancies were found in women with high endogenous LH. It is concluded that endometrial maturation is disturbed in women with low endogenous LH but can be rescued by mid-cycle stimulation of LH receptor with exogenous HCG in the absence of ovarian activity.