'Why have women not returned to use their frozen oocytes?': a 5-year follow-up of women after planned oocyte cryopreservation.

RESEARCH QUESTION: What are the reproductive choices and retrospective reflections of women at least 4 years after planned oocyte cryopreservation (POC)? DESIGN: This was an internet survey, using the REDCap application, of women who underwent POC, at a single-centre university-affiliated IVF unit, 4-8 years before the survey. The questionnaire addressed reproductive choices and outcomes following POC. RESULTS: Seventy-nine women who underwent POC during 2011-2014 were invited to participate, and 70 (89%) responded. Mean age at cryopreservation was 37.1 ± 2.4 (range 30-41) years, mean age at study participation 42.6 ± 2.6 (range 35-48) years, and mean time from first cryopreservation cycle to study participation 5.5 ± 1.3 (range 4-8) years. The main retrospectively reported reason for POC was not wanting to become pregnant without a partner (59, 84%). During the follow-up period, 44 women (63%) attempted to conceive either naturally or by assisted reproductive technology using fresh or cryopreserved oocytes. Of those, 28 women achieved a live birth (64% of those who tried to conceive). Fourteen respondents (20% of all respondents) reported using their cryopreserved oocytes, and three (21%) achieved a birth using those oocytes. Fifteen women (34%) of those who tried to conceive used donor spermatozoa. CONCLUSIONS: The most common reasons for not using frozen oocytes were achieving pregnancy without frozen oocytes or preferring not to have a child without a partner. A considerable proportion of women who had POC and were not interested in being a single parent by choice eventually try to conceive using donor spermatozoa several years later.

Ovarian follicular function is not altered by SARS-CoV-2 infection or BNT162b2 mRNA COVID-19 vaccination.

STUDY QUESTION: Does the immune response to coronavirus disease 2019 (COVID-19) infection or the BNT162b2 mRNA vaccine involve the ovarian follicle, and does it affect its function? SUMMARY ANSWER: We were able to demonstrate anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG in follicular fluid (FF) from both infected and vaccinated IVF patients, with no evidence for compromised follicular function. WHAT IS KNOWN ALREADY: No research data are available yet. STUDY DESIGN, SIZE, DURATION: This is a cohort study, composed of 32 consecutive IVF patients, either infected with COVID-19, vaccinated or non-exposed, conducted between 1 February and 10 March 2021 in a single university hospital-based IVF clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: A consecutive sample of female consenting patients undergoing oocyte retrieval was recruited and assigned to one of the three study groups: recovering from confirmed COVID-19 (n = 9); vaccinated (n = 9); and uninfected, non-vaccinated controls (n = 14). Serum and FF samples were taken and analyzed for anti-COVID IgG as well as estrogen, progesterone and heparan sulfate proteoglycan 2 concentration, as well as the number and maturity of aspirated oocytes and day of trigger estrogen and progesterone measurements. Main outcome measures were follicular function, including steroidogenesis, follicular response to the LH/hCG trigger, and oocyte quality biomarkers. MAIN RESULTS AND THE ROLE OF CHANCE: Both COVID-19 and the vaccine elicited anti-COVID IgG antibodies that were detected in the FF at levels proportional to the IgG serum concentration. No differences between the three groups were detected in any of the surrogate parameters for ovarian follicle quality. LIMITATIONS, REASONS FOR CAUTION: This is a small study, comprising a mixed fertile and infertile population, and its conclusions should be supported and validated by larger studies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to examine the impact of SARS-Cov-2 infection and COVID-19 vaccination on ovarian function and these early findings suggest no measurable detrimental effect on function of the ovarian follicle. STUDY FUNDING/COMPETING INTEREST(S): The study was funded out of an internal budget. There are no conflicts of interest for any of the authors. TRIAL REGISTRATION NUMBER: CinicalTrials.gov registry number NCT04822012.

Fertility preservation in reproductive-age women facing gonadotoxic treatments.

BACKGROUND: Advancements in the treatments for cancer and autoimmune and other hematologic conditions continue to improve survival and cure rates. Despite those changes, various gonadotoxic agents and other treatments can still compromise the future fertility of many women. Progress in medical and surgical reproductive technologies has helped to offset the reproductive consequences of the use of gonadotoxic therapies, and allows for future fertility and normal pregnancy. METHODS: A review of the literature was performed to outline the pathophysiology of gonadotoxicity from various treatments. The success of fertility preservation, fertility sparing, and cryopreservation options are reviewed. Barriers and facilitators to referral and oncofertility treatment in Canada are also outlined. RESULTS: According to the quality of the evidence, recommendations are made for fertility assessment, patient referral, cryopreservation, and other assisted reproductive technologies. CONCLUSIONS: To ensure ongoing fertility in women undergoing gonadotoxic treatments, assisted reproductive technologies can be combined with a multidisciplinary approach to patient assessment and referral.

Oncofertility in Canada: cryopreservation and alternative options for future parenthood.

BACKGROUND: Cancer can be a devastating diagnosis. In particular, malignancy and its indicated treatments have profoundly negative effects on the fertility of young cancer patients. Oncofertility has emerged as a new interdisciplinary field to address the issue of gonadotoxicity associated with cancer therapies and to facilitate fertility preservation. In Canada, these fertility issues are often inadequately addressed despite the availability of resources. The goal of this four-part series is to facilitate systemic improvements in fertility preservation for adolescent and young adult Canadians with a new diagnosis of cancer. METHODS: This article reviews fertility preservation options that use cryopreservation techniques. It also outlines some of the alternative options for future parenthood. RESULTS: Cryopreservation of a woman's gametes and gonadal tissue may involve embryo, oocyte, and ovarian tissue cryopreservation with or without ovarian stimulation. Similarly, male gametes and gonadal tissue may be cryopreserved. Techniques and success rates continue to improve. Third-party assistance through gamete donation, gestational carriers, and adoption are also alternative options for parenthood. CONCLUSIONS: Cryopreservation techniques are especially feasible options for fertility preservation in the newly diagnosed cancer patient.

Oncofertility in Canada: gonadal protection and fertility-sparing strategies.

BACKGROUND: Cancer can be a devastating diagnosis. In particular, malignancy and its indicated treatments have profoundly negative effects on the fertility of young cancer patients. Oncofertility has emerged as a new interdisciplinary field to address the issue of gonadotoxicity associated with cancer therapies and to facilitate fertility preservation. In Canada, fertility issues are often inadequately addressed despite the availability of resources. The goal of this four-part series is to facilitate systemic improvements in fertility preservation for adolescent and young adult Canadians with a new diagnosis of cancer. METHODS: Here, we review the fertility preservation measures currently available. Medical and surgical strategies are both outlined. RESULTS: Fertility-preserving strategies and gonadal protection have demonstrated variable success in a number of approaches. The value of hormone suppression is still in question for women. Progestins for endometrial cancer and alternative chemotherapies are other medical approaches. Gonadal shielding and protective surgical approaches have also been attempted. CONCLUSIONS: The techniques discussed here may be selectively considered and integrated into patient care in an attempt to preserve future fertility before initiating cancer treatment.

Oncofertility in Canada: an overview of Canadian practice and suggested action plan.

BACKGROUND: Cancer can be a devastating diagnosis. In particular, malignancy and its indicated treatments have profoundly negative effects on the fertility of young cancer patients. Oncofertility has emerged as a new interdisciplinary field to address the issue of gonadotoxicity associated with cancer therapies and to facilitate fertility preservation. In Canada, these fertility issues are often inadequately addressed despite the availability of resources. The goal of this four-part series is to facilitate systemic improvements in fertility preservation for adolescent and young adult Canadians with a new diagnosis of cancer. METHODS: Here, we describe the services currently available in Canada and the challenges associated with their utilization. Finally, we outline strategies to help maximize and facilitate fertility preservation in the young cancer patient. RESULTS: Despite an existing infrastructure to the oncofertility system in Canada, the ability of that system's components to function together and to coordinate patient care is a challenge. Areas of weakness include poor access and referral to fertility services, a lack of readily available education for patients and health care providers, and inconsistent interdisciplinary coordination in patient care. CONCLUSIONS: The implementation of a framework for multidisciplinary resource allocation, education, patient referral, and established lines of communication may facilitate a functional oncofertility system in Canada.

Oncofertility in Canada: the impact of cancer on fertility.

BACKGROUND: Cancer can be a devastating diagnosis. In particular, malignancy and its indicated treatments have profoundly negative effects on the fertility of young cancer patients. Oncofertility has emerged as a new interdisciplinary field to address the issue of gonadotoxicity associated with cancer therapies and to facilitate fertility preservation. In Canada, these fertility issues are often inadequately addressed despite the availability of resources. The goal of this four-part series is to facilitate systemic improvements in fertility preservation for adolescent and young adult Canadians with a new diagnosis of cancer. METHODS: In this article, we review the gonadotoxic effects of cancer treatment on young men and women of reproductive age. RESULTS: The detrimental effects of cancer on fertility can be severe and may vary depending on the chemotherapy, radiotherapy, or surgical treatments involved. CONCLUSIONS: Fertility preservation should be addressed in an effort to mitigate the gonadal damage that may come with cancer therapy.

Fertility preservation for cancer patients. Current options.

Prevalence of cancer has been increasing in the last years. Fortunately, during the last three decades, there has been a tremendous improvement in the success rates of cancer treatments and a continual rise in the survival rates. Given the improvement in survival rates with cancer treatment and developments in the field of reproductive medicine, fertility preservation for female cancer patients has become a very sensible issue. Today, several methods are available for preserving the reproductive potential of these patients. This review will focus on the options for fertility preservation offered to young cancer patients.

Working to eliminate multiple pregnancies: a success story in Québec.

In August 2010, the provincial government of Québec, Canada introduced funding of assisted reproduction treatment through the provincial health programme. Alongside this benefit, legislation was introduced to control assisted reproduction treatment activities in the province, including restrictions on the number of embryos that could be transferred in any one cycle. The aim of the programme was to transfer a single embryo in every cycle; multiple embryos could be transferred under suboptimal conditions but required physician justification. In the first 3 months of this programme, 1353 cycles of IVF were performed in five Québec assisted reproduction centres, with an overall clinical pregnancy rate of 32% per embryo transfer and 50% of transfers used elective single-embryo transfer (eSET). The multiple-pregnancy rate was only 3.7% per clinical pregnancy. In 2009, prior to the introduction of the programme, eSET was used in only 1.6% of embryo transfers, resulting in a multiple-pregnancy rate of 25.6%. These data demonstrate that providing provincially funded assisted reproduction treatment created an environment in which the aggressive use of eSET was not only possible, but also rapidly implemented. The result was a dramatic drop in multiple-pregnancy rates, approaching those for natural pregnancies.

Mechanisms of follicle selection and development.

Follicle recruitment and selection, the process that gives rise to the dominant follicle (DF) and the physiological state of the DF are important areas of research. The selection of a single ovarian follicle for further differentiation and finally ovulation is a shared phenomenon in monovulatory species including humans. The DF is different from other follicles because it can escape atresia (the fate of all other follicles). The DF cells develop corpus luteum if exposed to the luteinizing hormone (LH) surge. Several mechanisms for DF selection have been proposed. Rising follicle stimulating hormone (FSH) concentrations induce the emergence of a follicle wave and cohort attrition occurs during declining FSH concentrations, resulting in DF selection. Cohort secretions are initially responsible for the decline in FSH, which is subsequently suppressed by the selected DF lowering it below the threshold of FSH requirements of all other cohort follicles. The DF acquires relative FSH-independence in order to continue growth and differentiation during further declining FSH concentrations. A transition from FSH- to LH-dependence is postulated as the mechanism for the continued development of the selected DF. In addition, FSH and insulin-like-growth factor (IGF) enhance each other's ability to stimulate follicle cell function. Access of IGF II to their receptors is regulated by IGF binding proteins that are in turn regulated by specific proteases; all of which have been ascribed a role in DF development. One other shared mechanism recently proposed for DF selection is the possible differential regulation of blood vessel formation. Anti-Müllerian hormone (AMH) also plays a critical role in selection of the DF. AMH levels decline as the size of the follicle increases. Once follicles reach a size at which they are dominant, it has largely disappeared. From the time a follicle has been selected, the follicle destined to ovulate greatly enlarges and shows marked changes in its steroidogenic activity. LH surge causes a significant decline in gap junctions leading to dissociation of mural granulosa cells (GC) and expansion of the cumulus-oocyte complex (COC). The oocyte resumes its meiosis and progresses from prophase 1 to metaphase 2 at the time of ovulation. The concept of DF selection could be applied to the in-vitro maturation (IVM) program. Understanding the mechanism of DF selection in menstrual cycles is the key to planning the optimal timing of oocyte retrieval in order to obtain competent oocytes and embryos. Although the timing of oocyte retrieval is still open to debate, there is evidence to suggest that it may be better to retrieve oocytes before the small cohort follicles complete the process of atresia following selection of the DF.

Superior efficacy of enteric-coated mycophenolate vs mycophenolate mofetil in de novo transplant recipients: pooled analysis.

Dose reduction and discontinuation of mycophenolate mofetil (MMF) therapy because of gastrointestinal complications has been associated with increased risk of acute rejection episodes and graft loss. Enteric-coated mycophenolate sodium (EC-MPS) delays release of mycophenolic acid (MPA), and was designed to reduce MPA-related gastrointestinal adverse events. Data comparing the efficacy of EC-MPS vs MMF in de novo renal transplant (RTx) recipients from large prospective studies are limited. Therefore, a pooled data analysis was performed based on 1891 de novo RTx recipients receiving EC-MPS (n = 1289) or MMF (n = 602) plus cyclosporine and steroid therapy in 4 prospective multicenter studies with similar entry criteria. In all trials, the initial dose of EC-MPS was 1440 mg/d, and of MMF was 2000 mg/d; both dosages deliver equimolar amounts of MPA. Induction therapy was permitted in 2 studies per center practice. Multivariate logistic regression analysis was performed, adjusting other potential explanatory variables including recipient age, sex, and race/ethnicity; induction therapy; and diabetes mellitus at baseline. In addition, propensity scores were used to explain potential bias. Mean (SD) MPA dose (EC-MPS dosage was converted to MMF equivalent) during months 0 to 12 was similar: EC-MPS, 1820 (370) mg/d, vs MMF, 1860 (290) mg/d. However, at univariate and multivariate analyses, the rates of treatment failure, biopsy-proved acute rejection episodes, and graft loss were significantly lower with EC-MPS compared with MMF at month 12. In conclusion, this pooled analysis documents a substantial improvement in efficacy in de novo RTx recipients receiving EC-MPS vs MMF with concomitant cyclosporine and steroid therapy.

The effect of RANKL and OPG on bone mineral density in pre-dialysis chronic renal failure.

AIMS: The influence of pre-dialysis chronic kidney disease (CKD) on bone is ill defined. Isolation of specific pathogenic mechanisms would improve the understanding and therapeutic options. We therefore investigated whether parathyroid dysfunction, altered vitamin D and hormonal status, or RANKL and OPG have an influence on bone mineral density (BMD) in patients with pre-dialysis renal failure. MATERIAL: 132 patients with chronic renal failure stage 1 - 5 (not yet on dialysis) were investigated in a cross sectional study. Osteoprotegerin (OPG), receptor activator of nuclear factor kB ligand (RANKL), parathyroid hormone (whole, intact and 7-84 fragment), bone markers, sex hormones, and vitamin D status were assessed together with femoral neck and trochanter z-score. Correlation and multivariate analyses were performed between the different parameters and BMD. RESULTS: In the multivariate analysis a significant association was found between the femoral neck z-score and sRANKL (B = -0.45; p < 0.001), and OPG (B = 0.20; p < 0.05). A significant negative association was also found between the trochanter z-score and sRANKL (B = -0.32; p < 0.001). No associations were found between the trochanter z-score and OPG or the sRANKL/OPG ratio. The body mass index was the only additional marker associated with both FN z-score (B = 0.20, p < 0.05) and TR z-score (B = 0.20, p < 0.05). Neither markers of osteoblast nor osteoclast activity, or intact PTH, whole PTH, the PTH 7-84 fragment or vitamin D status were related to bone mineral density. CONCLUSION: Our results demonstrate that the RANKL/RANK/OPG system is associated with bone mineral density in pre-dialysis chronic renal failure.

Fertility preservation treatment for young women with autoimmune diseases facing treatment with gonadotoxic agents.

OBJECTIVE: To describe a case series of seven women with SLE and other systemic autoimmune rheumatic diseases (SARDs) who required cyclophosphamide therapy and underwent fertility preservation treatments. METHODS: Of the seven patients reported here, five women had SLE with nephritis, the sixth had immune thrombocytopenia purpura (ITP) and the seventh had microscopic polyangiitis (MPA) with renal involvement. All women were nulliparous and younger than 35 yrs. RESULTS: Patients with SLE underwent in vitro maturation (IVM) of immature oocytes aspirated during a natural menstrual cycle followed by vitrification of the matured oocytes if a male partner was not available, or vitrification of embryos if one was available. The patient with ITP and the patient with MPA underwent gonadotropin ovarian stimulation followed by oocyte or embryo vitrification. All women completed fertility preservation treatment successfully and mature oocytes or embryos (36 and 13, respectively) were vitrified. No complications were associated with this treatment and cytotoxic therapy was initiated as scheduled in all cases. CONCLUSIONS: Oocyte or embryo cryopreservation should be considered for fertility preservation in young women with SARDs who face imminent gonadotoxic treatment. In patients, where gonadotropin ovarian stimulation is deemed unsafe, IVM of immature oocytes, aspirated during a natural menstrual cycle, followed by vitrification or fertilization of the mature oocytes, seems to be safe and feasible. For patients in whom hormonal ovarian stimulation is not contraindicated, this method may be considered depending on the urgency to start cytotoxic therapy.

Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report.

We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.

Long-term administration of enteric-coated mycophenolate sodium (EC-MPS; myfortic) is safe in kidney transplant patients.

BACKGROUND: To date, there are no data on long-term use of enteric-coated mycophenolate sodium (EC-MPS; myfortic) from time of renal transplantation. We report the first long-term safety and efficacy data on EC-MPS when administered for up to 3 years post transplant. METHODS: De novo renal transplant recipients completing 1 year of treatment in a multicenter, randomized, double-blind trial of EC-MPS versus mycophenolate mofetil (MMF) were invited to take part in an open-label extension during which all patients received EC-MPS 720 mg b.i.d. Results from the period 12 - 36 months post transplant were compared to comparable data from MMF-treated patients taking part in two studies of everolimus versus MMF (RAD 201 and RAD 251). RESULTS: Of 367 patients completing the blinded core study, 247(62%) entered the open-label extension phase. During the first 24 months of the extension, the incidence, type and severity of adverse events were comparable between the newly-exposed and long-term EC-MPS patients. There were 2 deaths in the newly-exposed group and 4 among long-term EC-MPS patients, with 1 and 2 graft losses, respectively. Six patients (5%) in the newly-exposed group and 4 (3%) in the long-term EC-MPS group experienced biopsy-proven acute rejection. Cross-study comparisons indicated that the tolerability profile of EC-MPS was similar to MMF, including the incidence of adverse events, infections and malignancies, as was the incidence of efficacy events. CONCLUSION: These results demonstrate that EC-MPS with cyclosporine and steroids provides good long-term efficacy and tolerability, and confirm the safety of converting renal transplant patients from MMF to EC-MPS.

Changes in total parathyroid hormone (PTH), PTH-(1-84) and large C-PTH fragments in different stages of chronic kidney disease.

INTRODUCTION: Loss of renal function is accompanied by progressive increase in serum levels of intact parathyroid hormone (iPTH) in patients with end-stage renal disease (ESRD). There is a paucity of data regarding levels of PTH-(1-84) and its large carboxyl-terminal fragments (large C-PTH fragments) and progressive loss of kidney function in patients with chronic kidney disease (CKD). The current study was undertaken to describe the glomerular filtration rate (GFR)-dependent plasma concentrations of PTH-(1-84) and related large C-PTH fragments in adult patients with CKD by using different commercially available PTH assays. METHODS: We studied 80 Caucasian patients with CKD stages 1-5 without renal replacement therapy. Creatinine clearance was calculated by the Modification of Diet in Renal Disease (MDRD) formula. Levels of iPTH were determined by second-generation assays (iPTH Elecsys system, Roche Diagnostics; DUO total iPTH, Scantibodies Laboratory, Inc.; iPTH, Nichols Institute Diagnostics). Third-generation assays were used to measure PTH-(1-84) (CAP (cyclase activating PTH), Scantibodies; Bio-Intact PTH, Nichols). Levels of large C-PTH fragments and ratios of PTH-(1-84)/large C-PTH fragments were calculated and statistical analyses performed. RESULTS: Levels of iPTH and PTH-(1-84) showed CKD stage-dependent increases. Variations among the assays increased with progressive loss of kidney function. The assay from Scantibodies showed a GFR-dependent decrease of the ratio 1-84 PTH / large C-PTH fragment that was not observed with the Nichols assay. CONCLUSION: Increasing variations among the assays with progression of CKD emphasize the fact that the interpretation of measurements must take into consideration the specific assay. We found evidence for a possible preferential increase of the level of large C-PTH fragments over 1-84 PTH in a CKD stage-dependent manner (Scantibodies). The clinical implications of this finding have to be further evaluated by bone biopsy studies.

[Assisted reproduction technologies and the risk of fetal, chromosomal and genetic malformations].

BACKGROUND: Assisted reproduction techniques allowed thousands of otherwise infertile couples to attain pregnancy. As this technology moves into the mainstream of infertility treatment, it has become more critical to reassess its safety. OBJECTIVE: To review the birth outcome of patients undergoing conventional in-vitro fertilization and intracyto- plasmic sperm injection regarding fetal malformations, chromosomal and genetic abnormalities. METHODS: Selective review of the literature. RESULTS: Most of the published data is from observational studies and is not randomized or blinded. Unfortunately, most articles are inherently biased. Chromosomal and genetic abnormalities are increased probably only as a direct corollary to the underlying parental risk and not due to the technology itself. There is a slight increase in the congenital malformations rate, but inspection of these malformations reveal no clustering of any specific abnormality. CONCLUSIONS: Children born after assisted reproduction technologies have an increased risk of a major congenital malformation and chromosomal abnormalities compared with those born after natural conception. The risk is mainly due to paternal and maternal risk factors, which are more prevalent in couples who use assisted reproduction techniques for reproduction. Infertility-linked risk is highly probable for the observed findings. A technique-related risk, however, cannot be ruled out. Intracytoplasmic sperm injection appears to be a safe alternative for couples who otherwise would be unable to achieve pregnancy. The inherent risks associated with these genetically "at risk" couples mandate thorough evaluation and counseling before undertaking ICSI.

Human placental Hofbauer cells express sprouty proteins: a possible modulating mechanism of villous branching.

The development of the chorionic villous tree into a complex and organized ramified tubular network can be termed branching morphogenesis. Studying the molecular mechanisms involved in this process may contribute to the understanding of pregnancy complications such as preeclampsia. Sprouty (Spry) proteins are important regulators of branching morphogenesis and growth factor signaling. We analyzed the expression of Spry genes in human placenta. RT-PCR and immunohistochemistry were employed to detect placental Spry expression. Quantitative RT-PCR was used to assess the effect of FGF and reduced oxygen fraction on Spry gene expression. Spry 1, 2 and 3 expression was observed in placental tissue from all three trimesters. Our results reveal for the first time that Spry proteins are localized in the stroma of the chorionic villi, adjacent to cytotrophoblasts in areas of villous sprouting. Immunofluorescent double staining with anti-Spry and anti-CD68 confirmed that placental macrophages (Hofbauer cells) express Spry. Reduced oxygen fraction, FGF-4 and FGF-10 stimulated Spry-2 expression. Hofbauer cells also expressed c-Cbl, a protein that interacts with Spry. Placental expression of Spry and c-Cbl implies an important role for Hofbauer cells in placental development, possibly through a mesenchymal-epithelial interaction with trophoblasts. Regulation of Spry-2 expression by FGF-4 and FGF-10 suggests an orchestrated regulatory system that modulates villous branching.