S. Friedler

Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development

Abstract Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6–12 mm) were retrieved 34–36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

Very Low Sperm Count Affects the Result of Intracytoplasmic Sperm Injection

AbstractPurpose: The aim was to examine the influence of extremelylow sperm count on intracytoplasmic sperm injection(ICSI) outcome. Methods: Over 1000 consecutive unselected ICSI cycleswere divided into four groups according to spermconcentration of their patients: A, cryptozoospermia, 107 patients; B,sperm concentration of ≤ 1×104, 146 patients; C, spermcount of 1×104–1×105, 135 patients; and concentration of≤ 1×105 and < 10×106/ml (control group), 688 patients. Results: A significant decrease in pregnancy rate wasnoticed in the cryptozoospermic group in comparison to thecontrol group (20% vs. 31%). Fertilization rate in group Awas significantly lower in comparison to all other groups,respectively (46% vs. 52%, 54%, 61%). Embryo quality wasinferior in group A in comparison to the control group. Ahigher yet not statistically significant abortion rate wasobserved in the cryptozoospermic group (as well as in groupC) (30%, 27%) compared to the control group (15%). Conclusions: It seems that an extremely low sperm counthas a negative effect on the outcome of ICSI. Neverthelesspatients with cryptozoospermia should not be offered ICSItreatment with the ejaculated sperm before karyotype isestablished.

Surrogate in vitro fertilization outcome in typical and atypical forms of Mayer–Rokitansky–Küster–Hauser syndrome

BACKGROUND The genital malformations in Mayer-Rokitansky-Küster-Hauser syndrome (MRKH) are frequently accompanied by associated malformations whose forms were recently classified as typical (isolated uterovaginal aplasia/hypoplasia) and atypical (the addition of malformations in the ovary or renal system). The aim of this study was to compare the surrogate IVF performance of women with typical and atypical forms including their chances of achieving pregnancy. METHODS The follow-up data on a total of 102 cycles of surrogate IVF in 27 MRKH patients treated in our department between 2000 and 2010 were analysed. Twenty patients with the typical form who underwent 72 IVF cycles were compared with seven patients with the atypical form who underwent 30 IVF cycles. The various examined parameters of these intended mothers were age, hormonal profile during controlled ovarian hyperstimulation and laboratory outcome. RESULTS The mean number of gonadotrophin ampoules needed for stimulation and treatment duration was significantly higher in the atypical form (3600 ± 1297IU for 13 ± 2.3 days versus 2975 ± 967 IU for 11.6 ± 1.6 days, P≤ 0.01). Serum estradiol and progesterone levels measured on the hCG administration day were similar. A significantly higher mean number of follicles 12.6 ± 6 versus 8.9 ± 5.4, P≤ 0.03, metaphase II (MII) oocytes 8.7 ± 5.1 versus 6.7 ± 4.8, P≤ 0.05, fertilizations 6 ± 3.6 versus 4.4 ± 3.3, P≤ 0.03 and cleaving embryos 5.7 ± 3.8 versus 4.1 ± 3.3, P≤ 0.01 were available in patients with the typical form compared with those with the atypical form, respectively. There was no significant difference in fertilization rate, cleavage rate or the mean number of transferred embryos. Embryo quality of the transferred ones and pregnancy rate per cycle were also similar between the two groups. CONCLUSIONS Women with the typical form of MRKH needed fewer gonadotrophins and for a shorter duration for ovarian hyperstimulation. The mean number of follicles, oocytes, MII oocytes, fertilizations and cleaving embryos was higher among women with the typical form. Pregnancy rates were similar since the available number and quality of transferred embryos to the surrogate mother were not affected.

Presence of IL-18 in testicular tissue of fertile and infertile men.

Recently, IL‐18 was identified in human testes. Moreover, an inverse correlation was found between the levels of IL‐18 and the number and motility of spermatozoa. We examined the presence of IL‐18 protein in normal and impaired spermatogenesis. Testicular tissue specimens were taken from 25 nonobstructive azoospermic patients undergoing testicular sperm extraction and from autopsies of three healthy controls. The presence of IL‐18 in human testicular cells was examined by immunohistochemical staining of paraffin‐embedded sections, using a specific antibody for human IL‐18. In testicular tissue of healthy controls as well as in study cases, presence of IL‐18 was identified in somatic, mitotic, meiotic and post‐meiotic cells in correlation with their presence. In all patients, Leydig cells were less intensively stained. Mitotic cells were immunostained in the control group and less intensively in hypospermatogenesis and maturation arrest subgroups. Primary spermatocytes were in general most efficiently stained. The expression of IL‐18 mRNA (as examined by real‐time PCR analysis) showed significantly lower expression in testicular tissues with impaired spermatogenesis when compared to normal tissues. We report the first study demonstrating the presence of IL‐18 in human testicular tissue at the protein level. The presence of this cytokine in somatic as well as in different types of germ cells may suggest its involvement in the regulation of the spermatogenic process and steroidogenesis under physiological and pathological conditions.

Reproductive Performance of Patients Undergoing Intracytoplasmic Sperm Injection with 100% Implantation Rate

Purpose: To characterize the differences between twomatched groups of patients treated by ICSI: those pregnantafter all embryos transferred implanted (100% implantationrate) compared with nonpregnant patients. Methods: Twenty-one patients in whom one transferredembryo achieved a singleton pregnancy (group A) and 21pregnant patients to whom two or three embryos were transferredand achieved 11 twin and 10 triplet pregnancies(group B) compared with matched nonpregnant patients(group C and D, respectively). Results: The singleton pregnant patients were significantlyolder than the twin and triplet pregnancy patients. Althougha similar number of human menopausal gonadotropinampules were used in the singleton compared with the twinsand triplets a significantly lower number of oocytes andembryos were achieved at lower levels of estradiol on thehuman chorionic gonadotropin day in the former than inthe latter respectively. No difference was found between thepregnant women and their nonpregnant controls in any ofthe mentioned parameters. Good embryo morphology wasfound in 86% of the embryos in group A compared with62% in group C (P = 0.08) and 92% in group B comparedwith 66% in group D (P < 0.002). Conclusions: The only parameter in which pregnant patientswith 100% implantation rate differ from their nonpregnantcontrols was embryo quality.

Place des tests d’interaction sperme-mucus dans le bilan masculin de l’infécondité

ResuméIl est souvent nécessaire de faire une analyse fine du pouvoir fécondant des spermatozoïdes chez des couples infertiles. Le mucus cervical protège les spermatozoïdes et permet leur survie, assurant, à l’ovulation, une ascension continue de spermatozoïdes dans les voies génitales de la femme. Le test post-coïtal a longtemps été la seule épreuve fonctionnelle des spermatozoïdes. Il teste la pénétration et la survie des spermatozoïdes dans la glaire cervicale, les premieres d’une longue série d’étapes que doivent parcourir les gamètes mâles dans leur ascension vers l’ovule. Mais le TPC n’est pas dépourvu d’implications émotives et doit se faire le plus simplement possible pour éviter des inhibitions sexuelles. Dans les conditions optimales, le nombre de spermatozoïdes mobiles dans la glaire de l’endocol corrèle bien avec l’analyse du sperme et avec les chances de grossesses. En présence d’anticorps antispermatiques, on ne trouve au TPC que fort peu de spermatozoïdes en contraste avec la qualité de l’éjaculat ou beaucoup, mais immobiles ou agités. On peut aussi faire des tests in-vitro, (techniques de Kurzrok ou de Kremer) avec une glaire humaine ou avec des substituts bovins ou synthétiques. Ces épreuves évaluent bien la qualité du sperme mais ne corrèlent que partiellement avec le TPC naturel en raison de la nature complexe de la fonction du col utérin. Dans un groupe de FIV que nous avons analysé, le TPC corrèle avec les taux de fécondations et de grossesses en FIV et contribue à dépister le groupe à haut risque dans lequel des examens complémentaires peuvent trier les couples relevant de micro-injection. Cependant, le TPC a été critiqué pour sa méthodologie et surtout pour sa faible validité. Dans notre groupe de FIV, l’analyse de la validité du TPC et de tous les autres éléments d’appréciation de la qualité du sperme vis à vis des fécondations et grossesses confirme cette faible validité et montre qu’elle est commune à tous ces éléments d’appréciation, sans doute à cause de la nature multifactorielle du pouvoir fécondant des spermatozoïdes. L’analyse multifactorielle montre néanmoins que le TPC tient une place de premier plan parmi les examens pratiqués. Ainsi, l’évaluation du pouvoir fécondant des spermatozoïdes ne peut se fonder sur aucune épreuve isolée. Elle requiert une cascade judicieuse d’examens et c’est à leur lumière que l’on peut dresser un bilan valable et décider du geste thérapeutique rationnel.AbstractIn infertile couples, it may be necessary to finely analyze the fertilization ability of spermatozoa. The postcoital test (PCT) has long been the only sperm functional assay. It tests the sperm penetration and survival ability in the cervical mucus. These are the first steps of a long cascade of events that spermatozoa have to undergo during their ascension in the female genital tract, on their way to the site of fertilization. However, the PCT may evoke emotional stress and should be done in a simple way to avoid sexual inhibitions. In optimal conditions, the number of motile spermatozoa seen in the upper cervical mucus correlates well with semen analysis and the odds of subsequent pregnancies. Antisperm antibodies may impair the PCT and only few, if at all, or many immobile or shaking spermatozoa, may be seen in cervical mucus, in contrast to the sperm quality in the ejaculate. In vitro Sperm-Mucus tests may be done using Kurzrok or Kremer technique with human or bovine mucus as well as with synthetic media. These in vitro tests do allow a good evaluation of sperm quality but only partially correlate with natural PCT. In an IVF group the PCT correlates with IVF fertilization and pregnancy outcome. It helps detecting the high risk group in which additional tests, antisperm antibody detection, acrosome reaction test, hamster test (SPA), hemizona assay (HZA), are recommended. These tests may sort those couples requiring sperm egg micro-injection. However, the PCT has been criticized for its poor methodology and mainly for its weak validity. In this IVF group, we performed a validity analysis of PCT and all sperm tests against fertilization and pregnancy rate. It confirms the weak validity indices of PCT and of all sperm tests as well. Nevertheless, in the stepwise regression analysis of all these tests against IVF fertilization and pregnancy, F-test value of PCT was almost similar to SPA/HZA and higher than sperm morphology. Thus, PCT is important and the weak validity indices of all sperm tests are probably due to the multifactorial nature of sperm quality. So, no single test may reliably check sperm fertilization potential. Male work-up does require a cascade of examinations including PCT to allow reliable evaluation and rational therapeutic act.