Mammalian ovarian formation and differentiation takes place early in life, often before birth, but the ovary is not ready to fulfill its main purpose, that is, to ovulate a mature oocyte, until puberty. During early fetal development the germ cells populate the gonadal areas in close association with the mesonephros. The following developmental pattern of the ovary differs greatly among species, but one parameter is a must for all: each germ cell differentiates to an oocyte and becomes together with granulosa cells enclosed in a follicular entity. The pool of follicles is final and determines the length of the future reproductive lifespan. The finely tuned interaction between germ cells and somatic cells early in life is therefore crucial.

Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated.

The continued presence of stem cells and oogonia in the adult mammalian ovary Sir, We would like to comment on a recently published article (Byskov et al., 2011) that contradicts presence of germ stem cells or oogonia in the post-natal human ovary after the final clearing of these cells during the first 1 or 2 years of life.Presence of stem cells in adult ovary is a controversial area of research and needs to be resolved by the reproductive biologists.We have previously published data that choice of fixative and antigen retrieval protocols are crucial to detect Oct-4 positive cells in testicular tissues (Bhartiya et al., 2010).Similarly, Oosterhuis et al. ( 2011) have also recently reported that Oct-4 is an extremely sensitive and specific marker for human malignant germ cell tumors but may give falsenegative in biopsies fixed in Bouin's or Stieve's fixative.We consider that these may provide reasons that germ stem cells or oogonia might have been missed in the post-natal Bouin's fixed human ovary by Byskov et al.We request the group to refer to our recent publication (Parte et al., 2011) wherein we report that a gentle scraping of adult human, sheep, monkey and rabbit ovary surface releases stem cells in medium which can be characterized for various pluripotent markers by immunolocalization studies or by reverse transcription polymerase chain reaction.We continue to assert that stem cells do exist in adult ovaries and persist after menopause sets in.

La Folie des Grandeurs [Megalomania or Delusions of Grandeur](1962) painted by the Belgian surrealist René FG Magritte (1898-1967), is suggestive of the theory of ovist preformationism which held that unborn offspring existed fully formed in the eggs of its mother prior to conception. This idea was heralded by Antonio Vallisnieri (1661-1730), who even believed that in the ovary of the first woman, the entire human race in miniature was present from the beginning. In addition, Magritte’s painting is also reminiscent of the phenomenon of parthenogenesis, embryo development from an unfertilized egg, which is the biopathological origin of ovarian teratoma tumors. Oil on canvas. 100.4 x 81.3 cm. Access# 78-145 E. Photographer: Hickey-Robertson, Houston, TX, USA. Reproduced with kind permission from The Menil Collection, Houston TX, USA.

Human primordial germ cells (PGCs) can be recognized in the yolk sac wall, from 3-4 weeks post conception (wpc), in the hind gut epithelium from week 4 and in the gonadal area from early week 5. The objective of this study was to map the migration route of PGCs and elucidate the role of the nervous system in this process. Sixteen human specimens, 5-14 wpc obtained from legal abortions were included. On serial paraffin sections, PGCs were detected immunohistochemically by expression of OCT4 and c-Kit, nerve fibers by β-III-tubulin and stem cell factor (SCF) as a possible chemoattractive cue for PGC migration. PGCs were present in the hind gut epithelium, in the mesenchyme of the dorsal mesentery and in the developing gonadal ridge of 4-6 wpc embryos, prior to connections between the enteric and the sympathetic nervous system. From 6 wpc onwards, the PGCs travelled along the developing nerve fibers from the wall of the hind gut via the dorsal mesentery to the midline of the dorsal wall and laterally into the gonads. Numerous PGCs were still present in the nervous system by 14 wpc. PGCs in 4-5 wpc embryos are suggested to leave the gut epithelium by EMT-like transition. SCF may facilitate further migration, but after establishment of connections between the enteric and sympathetic nervous systems. PGCs follow sympathetic nerve fibers towards the gonads. PGCs failing to exit the nerve branches at the gonadal site, may continue along the sympathetic trunk ending up in other organs where they may form germ cell tumors if not eliminated by apoptosis.

Objective: The present study was performed to evaluate whether the hormone profiles of the mid-luteal phase impact on the chances of conceiving during the following menstrual cycle in connection with intrauterine insemination (IUI) treatment. Design: Observational clinical study. Setting: Infertility clinic. Patient(s): 92 women underwent a total of 288 IUI treatment cycles, without use of exogenous hormones. Intervention(s): The mid-luteal hormone profiles including levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, oestradiol, progesterone, androstenedione and testosterone, were measured in the preceding cycle of IUI treatment. Main outcome measure(s): The mid-luteal hormone profiles were correlated to whether the women conceived in the following natural menstrual cycle in which IUI treatment was performed and to whether ovulation occurred on the same ovary in two consecutive cycles (ipsilateral ovulation) or jumped from one ovary to the other (contralateral ovulation). Result(s): When ovulation occurred from the same ovary in two consecutive cycles mid-luteal levels of progesterone of the preceeding cycle were significantly lower in conceptional than non-conceptional cycles. No significant differences of LH, FSH, estradiol, androstendione and testosterone were observed when correlating the different parameters. Conclusion(s): Lower mid-luteal progesterone levels seem to enhance chances of conception when ovulation occurs in the same ovary for two consecutive cycles.

Identification of the ovary at the time of ovulation during three consecutive menstrual cycles results in one of eight ovulation patterns, left-left-right, right-left-right, left-right-right, and right-right-right of right-sided ovulation and right-right-left, left-right-left, right-left-left, and left-left-left of left-sided ovulation. Our data suggest that IVF and IUI treatment in cycles in which development of the preovulatory follicle(s) occurs in the right-sided ovary-and ovulations took place from the left-sided ovary in the preceding two cycles (left-left-right)-is likely to show the best pregnancy potential and high offspring sex ratio.

Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri- and post-natal ovaries.For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes.Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles.We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla.

OBJECTIVES:The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time- and labour-consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G(2)+M (SG(2) M) cell-cycle phases as indicators of cell proliferation.METHODS:Cell suspensions from 35 human embryonic gonads at days 37 to 68 post-conception (pc) were immunomagnetically sorted into C-KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG(2) M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads.RESULTS:During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG(2) M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation.CONCLUSIONS:Cell proliferation as indicated by S and SG(2) M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.

The present study assessed whether the smoking habits of fathers around the time of conception affected the period in which daughters experienced menstrual cycles (i.e., the reproductive life span). The study revealed that the smoking habits of the farther shortened the daughters' reproductive life span compared with daughters whose fathers did not smoke.

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

BACKGROUND The number of germ cells in human embryonic and fetal ovaries in relation to age is currently based on volumetric estimations from one study including a total of 12 ovaries. Six recent publications present stereological estimations of the number of germ cells in ovaries and testes for the first two trimesters. METHODS Germ cell numbers from 103 human first and second trimester gonads aged 37-133 days post-conception (p.c.), obtained after legal termination of pregnancy, were collected from six independent studies that all used similar validated stereological methods for estimating germ cell numbers as well as somatic cell numbers. RESULTS Statistically, the six studies estimated similar number of germ cells (P > 0.05) and no interaction between the studies and age was found (P > 0.05), indicating that the increase in cell numbers in relation to age was of comparable magnitude in each study. The number of germ cells increased from a mean of 7200 to 4,933,000 in fetal ovaries and from 3700 to 1,417,000 in fetal testes, from week 5 to week 19 p.c. A higher rate of increase was found for female germ cells as compared with males (P = 0.004). During the same period, the number of somatic cells increased from a mean of 158,000 to 1,017,000 in ovaries and from 154,000 to 2,035,000 in testes, respectively. CONCLUSIONS By the use of validated stereological methods, this study provides more accurate and improved information on human germ and somatic cell numbers in ovaries and testes during the first two trimesters of pregnancy.

A.G. Byskov1,*, P.E. Høyer2†, C. Yding Andersen1, S.G. Kristensen1, A. Jespersen3, and K. Møllgård2 Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Rigshospitalet, Section 5712, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Department of Cellular and Molecular Medicine, Developmental Biology Unit, The Panum Institute, Copenhagen, Denmark Laboratory of Zoology, Biological Institute, University of Copenhagen, Copenhagen, Denmark

Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR.

BACKGROUND: Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri-and post-natal ovaries. METHODS: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. RESULTS: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. CONCLUSIONS: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla.

BACKGROUND: Early menarcheal age is a risk factor for breast and ovarian cancers and is also associated with an increased spontaneous abortion rate. The aim of this study was to investigate whether there is a link between early menarcheal age and the offspring sex ratio.METHODS: We recorded the sex of 21 208 live born infants, all singletons, born to 10 847 premenopausal women (mean attending age: 37.5 +/- 7.2 years, range 22-54) who attended our clinics for obstetrical and gynaecological assessment. We calculated the sex ratio of newborn infants in relation to the mothers' age of menarche (from 9 to 18 years) and to the number of infants per woman (i.e. fertility index).RESULTS: A low offspring sex ratio (males/females) of 0.800 was observed in mothers who entered menarche at the age of 9 years; the odds ratio (OR) (95% confidence intervals) compared with those of control group with menarche at age 14 was 0.72 (0.29-1.79). The sex ratio remained low, until the group of mothers who entered menarche at 12 years of age [1.009; OR: 0.90 (0.85-0.95)]. The highest sex ratio was observed in mothers with a menarcheal age of 14 years (1.118; OR: 1.00 control) and it gradually decreased until the menarcheal age of 17 years [1.000; OR: 0.89 (0.15-5.26)]. The fertility index was also low in women with menarche at the age of 9 to 11 years.CONCLUSIONS: Women entering menarche outside the normal range, especially those with earlier menarche, may have an increased chance of producing female offspring.

Sir, We read with interest the article by Fukuda et al. (2011). The title ‘The sex ratio of offspring is associated with the mothers’ age at menarche’ suggests a direct connection between age at menarche and sex of offspring. We consider this interpretation a stretch of the presented data and want to comment on some issues in their study. Additionally, results from our substantially larger Danish cohort are presented. We would like to start with raising some points in the analysis by Fukuda et al. (2011) that make their results look more extreme than they actually are:

BACKGROUND Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimester gonads in relation to maternal smoking. METHODS The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated stereological methods were used to estimate gonadal cell numbers in histological sections. Results were also evaluated in the context of previously published data on ovaries from our laboratory. RESULTS A significant reduction in the number of germ cells by 55% [95% confidence interval (CI) 74-21% reduction, P = 0.004] and somatic cells by 37% (95% CI 59-3%, P = 0.023) was observed in testes prenatally exposed to maternal cigarette smoking, compared with unexposed. The effect of maternal smoking was dose-dependent being higher in the heavy smokers and remained consistent after adjusting for possible confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS Prenatal exposure to maternal cigarette smoke reduces the number of germ and somatic cells in embryonic male and female gonads. This effect may have long-term consequences on the future fertility of exposed offspring. These findings may provide one potential cause of the reduced fertility observed during recent years.