Lucette A.J. van der Westerlaken

Piwi Proteins and piRNAs in Mammalian Oocytes and Early Embryos

Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi and piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, whereas those in bovine ovary only express PIWIL1. In human, macaque, and bovine ovaries, we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis, we show that these maturing oocytes strongly and specifically express the PIWIL3 protein, alongside other, known piRNA-pathway components. A piRNA pool is still present in early bovine embryos, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal that there are highly dynamic piRNA pathways in mammalian oocytes and early embryos.

Evaluation of Pregnancy Rates After Intrauterine Insemination According to Indication, Age, and Sperm Parameters

Purpose:Our purpose was to evaluate intrauterine insemination results obtained in our clinic and identify prognostic factors for the chance of pregnancy.Methods:A retrospective study of data from 1989 to 1996 was undertaken. Only first attempts were included in this study, except for the part on the cumulative pregnancy rates. Couples with either one-sided tubapathology, hormonal dysfunction, idiopathic infertility, or andrological indication were selected. All women were stimulated with clomiphene citrate. Five hundred sixty-six couples who underwent 1763 cycles were included in the study.Results:The overall pregnancy rate for first pregnancies was 6.9% per cycle and 21.4% per patient. For first intrauterine insemination attempts this was 8.8% per cycle/patient, varying between 5.0% for andrological indication and 10.6% for tubapathology, 10.0% for idiopatic indication, and 10.3% for hormonal indication. These differences were not significant. Age did not have a significant effect either, although there were no pregnancies observed in women 40 years or older. The number of inseminated spermatozoa significantly affected the pregnancy rate: <2 million, 4.6%; ≥2 to <10 million, 3.9%; and ≥10 million, 11.3%.Conclusions:Unless semen characteristics are insufficient, intrauterine insemination is a useful treatment for infertile couples.

Perinatal outcome, health, growth, and medical care utilization of 5-to 8-year-old intracytoplasmic sperm injection singletons

OBJECTIVE To evaluate short- and long-term health in intracytoplasmic sperm injection (ICSI) singletons. DESIGN Follow-up study. SETTING University medical center, assessments between March 2004 and May 2005. PATIENT(S) Singletons born between June 1996 and December 1999 after ICSI in the Leiden University Medical Center laboratory were compared with matched singletons born after IVF and natural conception. INTERVENTION(S) Mode of conception. MAIN OUTCOME MEASURE(S) An examiner blinded to the conception mode of the child assessed congenital malformations and growth. Information on pregnancy, perinatal period, birth defects, general health, and medical consumption was obtained through questionnaires. RESULT(S) Outcomes of children conceived by ICSI and IVF (n = 81/81, preterm infants excluded) were comparable or even more positive for ICSI. Perinatal outcomes were poorer after ICSI than natural conception: prematurity: P=.014; low birth weight: odds ratio = 7.4, 95% confidence interval (CI) [0.9; 62.5]; mean birth weight: Delta = 186 g, 95% CI [21; 351]. The ICSI mothers had more pregnancy complications (n = 33 vs. 18) and in-hospital deliveries (prevalence ratio 1.36, 95% CI 1.17; 1.48). No further differences were found between ICSI and natural conception children on congenital malformations, health, growth, and medical consumption (n = 87/85, preterm infants included). CONCLUSION(S) No adverse health outcomes were identified in ICSI singletons up to age 5-8 years compared to IVF and natural conception singletons, besides poorer perinatal outcomes after ICSI versus natural conception.

Development of the follicular basement membrane during human gametogenesis and early folliculogenesis

BackgroundIn society, there is a clear need to improve the success rate of techniques to restore fertility. Therefore a deeper knowledge of the dynamics of the complex molecular environment that regulates human gametogenesis and (early) folliculogenesis in vivo is necessary. Here, we have studied these processes focusing on the formation of the follicular basement membrane (BM) in vivo.ResultsThe distribution of the main components of the extracellular matrix (ECM) collagen IV, laminin and fibronectin by week 10 of gestation (W10) in the ovarian cortex revealed the existence of ovarian cords and of a distinct mesenchymal compartment, resembling the organization in the male gonads. By W17, the first primordial follicles were assembled individually in that (cortical) mesenchymal compartment and were already encapsulated by a BM of collagen IV and laminin, but not fibronectin. In adults, in the primary and secondary follicles, collagen IV, laminin and to a lesser extent fibronectin were prominent in the follicular BM.ConclusionsThe ECM-molecular niche compartimentalizes the female gonads from the time of germ cell colonization until adulthood. This knowledge may contribute to improve methods to recreate the environment needed for successful folliculogenesis in vitro and that would benefit a large number of infertility patients.

On the development of extragonadal and gonadal human germ cells

ABSTRACT Human germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception). Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development. Summary: The presence and developmental trajectory of ectopic human germ cells in the adrenals and gonads during the first and second trimesters is investigated by comparing the expression dynamics of early, late and meiotic germ cell markers.

Human Extravillous Trophoblasts Penetrate Decidual Veins and Lymphatics before Remodeling Spiral Arteries during Early Pregnancy

In humans, the defective invasion of the maternal endometrium by fetal extravillous trophoblasts (EVTs) can lead to insufficient perfusion of the placenta, resulting in pregnancy complications that can put both mother and baby at risk. To study the invasion of maternal endometrium between (W)5.5–12 weeks of gestation by EVTs, we combined fluorescence in situ hybridization, immunofluorescence and immunohistochemistry to determine the presence of (male) EVTs in the vasculature of the maternal decidua. We observed that interstitial mononuclear EVTs directly entered decidual veins and lymphatics from W5.5. This invasion of decidual veins and lymphatics occurred long before endovascular EVTs remodelled decidual spiral arteries. This unexpected early entrance of interstitial mononuclear EVTs in the maternal circulation does not seem to contribute to the materno-placental vascular connection directly, but rather to establish (and expand) the materno-fetal interface through an alternative vascular route.

At Term, XmO and XpO Mouse Placentas Show Differences in Glucose Metabolism in the Trophectoderm-Derived Outer Zone

Genetic mouse model (39,XO) for human Turner Syndrome (45,XO) harboring either a single maternally inherited (Xm) or paternally inherited (Xp) chromosome show a pronounced difference in survival rate at term. However, a detailed comparison of XmO and XpO placentas to explain this difference is lacking. We aimed to investigate the morphological and molecular differences between XmO and XpO term mouse placentas. We observed that XpO placentas at term contained a significantly larger area of glycogen cells (GCs) in their outer zone, compared to XmO, XX, and XY placentas. In addition, the outer zone of XpO placentas showed higher expression levels of lactate dehydrogenase (Ldha) than XmO, XX, and XY placentas, suggestive of increased anaerobic glycolysis. In the labyrinth, we detected significantly lower expression level of trophectoderm (TE)-marker keratin 19 (Krt19) in XpO placentas than in XX placentas. The expression of other TE-markers was comparable as well as the area of TE-derived cells between XO and wild-type labyrinths. XpO placentas exhibited specific defects in the amount of GCs and glucose metabolism in the outer zone, suggestive of increased anaerobic glycolysis, as a consequence of having inherited a single Xp chromosome. In conclusion, the XpO genotype results in a more severe placental phenotype at term, with distinct abnormalities regarding glucose metabolism in the outer zone.