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The introduction of intracytoplasmic sperm injection (ICSI) provided an effective treatment for infertile couples whose infertility was attributed to male factors. However, some of them face poor results after ICSI and subsequently use artificial insemination with donor sperm (AID). Only a few studies have reported on the clinical outcome of AID cycles after previous failed ICSI cycles, with contrasting results. The results reported here involve a cohort of 47 couples undertaking 175 AID cycles after 120 failed ICSI cycles for various reasons. Couples were allocated to two groups according to the availability of top quality embryos (TQE) in ICSI cycles. In our series, AID was successful for couples with and without TQE previously transferred in ICSI cycles, the live birth rate (LBR) per cycle being 20.0% and 13.3%, respectively. However, couples with TQE tended to succeed more rapidly than couples with poor quality embryos, with a higher cumulative LBR (68.0% versus 54.5%, respectively). These findings demonstrate that even couples with a history of unsuccessful ICSI cycles because of poor embryo quality are able to achieve high LBR after AID cycles. However, such couples have a lower cumulative LBR and are required to be more patient to achieve parenthood.

Preimplantation embryo development is one of the key features with implantation itself to achieve a pregnancy. Assisted Reproductive Technologies both in human and animal have improved our knowledge on these events, although it remains elusive to predict embryo potential to give a baby. Among various ways to define embryo viability, noninvasive approaches get a serious advantage linked to the final transfer of the embryo. Techniques devoted to characterize the embryo secretome using proteomic or metabolomic approaches may be non invasive. Based on a direct identification of products of the embryo metabolism or an assessment of profile(s) related with embryo viability, they have greatly improved their sensitivity to allow their use in clinical embryology, once validated. Oocyte-cumulus dialogue, as a key factor for oocyte competence to meiosis and embryo development, was particularly concerned with both genomic and proteomic assessment of cumulus cells. While it is not possible to designate at the time being which among these approaches will be robust and cost-efficient enough to help routinely the clinical embryologist in assisted reproductive techniques (ART), one can predict that our ability to select the "right" embryo will combine morphological criteria already available with validated biomarkers.

Summary The effect of human follicular fluid (hFF) on the cholesterol and phospholipid content and the movement characteristics of human spermatozoa were studied. Semen was selected by a discontinuous Percoll gradient and incubated during in vitro capacitating conditions with B2 medium supplemented with hFF 20%. Percoll pelleted spermatozoa were incubated in either B2 (B2-Percoll) or B2 supplemented with hFF (hFF-Percoll). In hFF-Percoll, we observed a time-dependent (24 h) decrease in both the cholesterol and phospholipid contents (cholesterol: 10.1 vs. 8.7 nmol 10-7 spermatozoa; phospholipids: 17.5 vs. 15.7 nmol 10-7 spermatozoa, P < 0.05). This decrease in cholesterol and phospholipids in human spermatozoa was concomitant with a high straight line velocity, a high progressive motility percentage and an increased value of lateral head displacement without any significant alteration of the spermatozoal membrane. No modification of the cholesterol: phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll (0.61, 0.54) in hFF-Percoll (0.59, 0.63) was observed when compared with original control semen. It is suggested that the decrease in cholesterol and phospholipids in hFF-Percoll may be taken into account for the changes of membrane modification as part of the capacitation process.

A cohort follow-up study was designed to compare the efficacy of IVF using frozen donor semen (IVF-D) following previously failed DI cycles (unexplained female infertility) and direct IVF-D treatment because of tubal infertility (control group). The cohort comprised 189 couples initiating IVF-D after previously failed DI cycles (n = 126) or directly (n = 63). Couples were followed until completion (success or drop-out for personal or medical reasons). Live births and drop-out were expressed both as rate per cycle and crude cumulative rate. Characteristics of IVF-D cycles were similar between the two groups. Moreover, overall outcome was also similar in terms of crude cumulative live birth rate (54.0 versus 57.1% for failed DI cycles and tubal infertility groups respectively). This is the first report on crude cumulative live birth rate based on a cohort follow-up study in unexplained previously failed DI cycles and tubal infertility. Previously failed DI cycles did not impair the chances of achieving a successful pregnancy using IVF-D in this series. Slight oocyte dysfunction, which might underlie the failure of DI cycles, might be overcome using IVF-D.

The need to assess the health of children born after assisted reproductive technologies (ART) using frozen donor spermatozoa has been a major concern for the last decade. Most available published studies are confusing (since they are retrospective) and refer to small numbers of cases or involve a bias. Two prospective and large population-based studies have been published, one in France and the other in Australia. The general characteristics of children born after ART using frozen spermatozoa was unchanged in comparison with the general population (weight, prematurity, stillbirths, sex ratio). However, the rate of multiple pregnancy was increased, first in artificial insemination with donor semen (AID), where the use of hormonal treatment may be too heavily prescribed, and second in IVF with donor semen (IVF/D) where both the ovulation induction treatments and embryo transfer policy increase the rate of multiple pregnancies. As far as birth defects or chromosomal abnormalities are concerned, no difference has been observed from the general population. Finally, the use of frozen spermatozoa does not seem to affect the health of children conceived by AID or IVF/D. The psychosocial development of such infants is not as well known because of the confidentiality in many countries concerning sperm donation. However, the available information on the psychosocial development of these children up to the age of 8-10 years appears to be reassuring. Larger and longer studies are needed to answer such questions, particularly with regard to adulthood.

In order to evaluate the role of human follicular fluid (HFF) on the fertilizing capacity of spermatozoa, we studied the effect of HFF on the lipid composition and on the movement characteristics of human spermatozoa. Spermatozoa (spz) from normospermic patients were prepared with a discontinuous Percoll gradient and incubated in Menezo B2 medium with or without a supplement of 20% HFF (HFF-Percoll spz and B2-Percoll spz respectively) for 2 and 24 h. After 2 h HFF incubation, percentage progressive motility, straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were improved in HFF-Percoll spz as compared to B2-Percoll spz (P < or = 0.05). After a longer incubation period (24 h), lipid changes appeared in HFF-Percoll spz with lower levels of cholesterol (P = 0.02) and phospholipids (P = 0.05). No modification of the cholesterol/phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll spz or HFF-Percoll spz was observed. Such decreases in lipid content of HFF-Percoll spz may be factors which could be taken into account as constituting part of membrane modifications during the capacitation process.

Summary A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing -thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.

IntroductionLes femmes en attente de don d’ovocytes sont confrontées à diverses difficultés peu explorées dans la littérature : vécu subjectif douloureux, échecs des FIV précédentes, long délai d’attente d’un don, dissociation de la filiation génétique et gestationnelle… L’objectif principal est de déterminer si l’« attente d’un don d’ovocytes » est anxio-dépressiogène, et si le locus de contrôle externe constitue un facteur protecteur. MethodsLes 3 groupes de femmes inclus sont en attente d’un don d’ovocytes (n = 10), en cours d’une première FIV (FIV1 ; n = 34) ou d’une deuxième FIV ou plus (FIV2+ ; n = 39). Elles ont été évaluées sur l’anxiété (State-Trait Anxiety Inventory), la dépression (Beck Depression Inventory), les locus de contrôle, la réaction à l’infertilité. RésultatsLes femmes receveuses étaient significativement plus âgées. Elles étaient significativement plus anxieuses mais pas plus dépressives que les groupes FIV. Leur anxiété actuelle était en partie expliquée par une internalité plus marquée. Pourtant, chez les femmes infertiles en général, c’est l’externalité de type « Autre toutpuissant » qui expliquait en partie la diminution de l’anxiété et la diminution du vécu négatif émotionnel de l’infertilité. ConclusionCes résultats sur le locus de contrôle des femmes infertiles éclairent la réflexion sur le mode relationnel médecin malade, chez des patientes anxieuses sans maîtrise de leur fécondité et devant faire confiance aux équipes soignantes.

Both the ability to freeze human spermatozoa and the possibility of pregnancy following intrauterine insemination have existed for >40 years. There have been a number of improvements during that time concerning the methods of freezing and thawing human spermatozoa. Initially, the use of the cryoprotective properties of glycerol allowed a major improvement; subsequently, changes were mainly empirical. It was a long time before specific cryobiological studies were undertaken. However, the necessity for these became apparent with the partial recovery or sometimes loss of motility after freezing either subfertile semen before chemotherapy or radiotherapy, or spermatozoa collected from non-physiological situations (epididymal or testicular spermatozoa). The main trends in improvement have defined end-points other than the percentage of motility recovery or the assessment of ultrastructural damage. More sensitive criteria of the objective assessment of motility, energy status, damage to the plasma membrane or to subcellular elements, chromatin stability and chromosomal damage have been proposed as complementary end-points to better assess sperm cryopreservation. A different approach was related to the biochemical environment and physical conditions imposed on spermatozoa during the freezing and thawing process. Biochemical changes were assessed following different combinations of various extenders which attempted either to better preserve some parameter or to avoid the tendency towards drastic increase in osmotic pressure. Analysis of physical conditions was linked to the rate of cooling, freezing and warming, and was based on cryobiological studies. Finally, even though such improvements are not negligible, many questions remain unanswered. The extensive use of frozen spermatozoa during assisted reproductive techniques, together with the development of assisted fertilization using surgically collected spermatozoa, creates the need for additional studies to improve the cryopreservation of human spermatozoa.

The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [14C]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were approximately 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 microM progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (approximately 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (approximately 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.

This study measured glycosaminoglycans (GAGs) in human follicular fluid (hFF) obtained from patients undergoing hormonal stimulation with combined GnRH agonist followed by gonadotropin hormone (hMG or FSH) for in vitro fertilization (IVF). The GAGs were partially characterized through the use of various mucopolysaccharidases and then their effects tested on human sperm motility. In hFF, the GAG and protein concentrations were 4.4 +/- 1.3 mg/L and 32.6 +/- 3.2 g/L respectively. Chondroitins (CS) and hyaluronic acid (HA) significantly stimulate sperm motion in comparison to the control. The oocytes inseminated with GAGs-pretreated spermatozoa showed a significantly higher rate of cleavage and pregnancy or delivered number. The present results suggest that hFF is a rich source of sulfated glycosaminoglycan (CS), and non-sulfated glycosaminoglycan (HA). The stimulatory effects of hFF on human sperm motility may well depend on CS and HA. GAGs pretreated sperm may enhance the fertilizing ability of spermatozoa and thus the IVF outcome.

Functional ultrastructural changes in human sperm heads were evaluated following semen dilution in cryoprotective medium (with and without seminal plasma) as well as freezing/thawing. Plasma membranes were as much altered by dilution as by freezing/thawing when compared with fresh samples. The most striking effect on acrosome noted during freezing/thawing was a dramatic decline in percentage of intact spermatozoa. Acrosomal changes seemed to be less important in the fractions without seminal plasma. These deleterious effects were very demonstrable using transmission electron microscopy (TEM), rather than conventional staining. TEM is useful in obtaining more detailed information on membrane and acrosome integrity until a specific procedure to evaluate functional/physical integrity can be found. Cyropreservation in the absence of seminal plasma can be used for intrauterine insemination in AIH, AID programs with frozen semen.

Studies in animals have highlighted a possible role for growth factors, particularly IGF-I on cellular replication and cytodifferentiation in the ovary. At this time, few studies have been performed about IGF-I in the human ovary. From 38 women undergoing in Vitro Fertilization 293 antral fluids were collected and assessed for steroids (estradiol and progesterone), FSH and IGF-I. Two induction treatments were compared: clomiphene citrate/hMG (group A, N = 15), triptoreline/hMG (group B, N = 23). We also studied relationships between quantitative parameters and oocyte collection or oocyte corona cumulus complex maturity. In group B, the highest antral estradiol levels were found in follicles yielding an oocyte (p

Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.