The history of Belgian assisted reproduction technology cycle registration and control: a case study in reducing the incidence of multiple pregnancy.

STUDY QUESTION: What is the effect of a legal limitation of the number of embryos that can be transferred in an assisted reproductive technology (ART) cycle on the multiple delivery rate? SUMMARY ANSWER: The Belgian national register shows that the introduction of reimbursement of ART laboratory costs in July 2003, and the imposition of a legal limitation of the number of embryos transferred in the same year, were associated with a >50% reduction of the multiple pregnancy rate from 27 to 11% between 2003 and the last assessment in 2010, without any reduction of the pregnancy rate per cycle. WHAT IS KNOWN ALREADY: Individual Belgian IVF centres have published their results since the implementation of the law, and these show a decrease in the multiple pregnancy rate on a centre by centre basis. However, the overall national picture remains unpublished. STUDY DESIGN, SIZE, DURATION: Cohort study from 1990 to 2010 of all ART cycles in Belgium (2685 cycles in 1990 evolving to 19 110 cycles in 2010), with a retrospective analysis from 1990 to 2000 and prospective online data collection since 2001. PARTICIPANTS/MATERIALS, SETTING, METHODS: Registration evolved from paper written reports per centre to a compulsory online registration of all ART cycles. From 2001 up to mid-2009, data were collected from Excel spread sheets or MS Access files into an MS Access database. Since mid-2009, data collection is done via a remote and secured web-based system (www.belrap.be) where centres can upload their data and get immediate feedback about missing data, errors and inconsistencies. MAIN RESULTS AND THE ROLE OF CHANCE: National Belgian registration data show that reimbursement of IVF laboratory costs in July 2003, coupled to a legal limitation in the number of embryos transferred in utero, were associated with a 50% reduction of the multiple pregnancy rate from 27 to 11% without reduction of the pregnancy rate per cycle, and with an increase in the number of fresh and frozen ART cycles due to improved access to treatment. LIMITATIONS, REASONS FOR CAUTION: There is potential underreporting of complications of ART treatment, pregnancy outcome and neonatal health. WIDER IMPLICATIONS OF THE FINDINGS: Over the 20 years of registration, the pregnancy rate has remained constant, despite the reduction in the number of embryos transferred, optimization of laboratory procedures and stimulation protocols, introduction of quality systems and implementation of the EU Tissue Directive over the period 2004-2010. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study. None of the authors has any conflict of interest to declare.

Trends in embryo disposition decisions: patients' responses to a 15-year mailing program.

BACKGROUND: This study examined the responses of patients of a Belgian fertility center to mailed requests to make or renew an embryo disposition decision (EDD), over a period of 15 years, to investigate trends in the decisions. METHODS: A retrospective analysis was performed on a mailing program from 1992 to 2006, for patients, of the Department of Reproductive Medicine, Ghent University Hospital (Belgium), from whom embryos had been cryopreserved at least 2 years. RESULTS: In 15 years, 3840 EDD forms were prepared for 2334 couples or female patients. The number of forms increased from 21 in 1992 to 558 in 2006. Each year, around a third of the forms were not returned. In general, a quarter of patients who received more than one form never answered. Donation to others for reproduction was overall the least popular option and decreased over the years. The rising trend in decisions to discard reversed into a negative trend from the introduction of donation for science (1997). Since then, donation for science has been the most popular option and its popularity increased with time. In 15 years, 2504 embryos were donated for science. More than a quarter of the patients who chose more than one final EDD in different years did not select the same EDD the second time. CONCLUSIONS: This study showed a positive trend in donation for science and a negative trend in donation to others and discarding. A substantial number of individual patients chose different types of EDDs in consecutive mailings, which shows that advance EDD directives should be used with caution.

To continue or discontinue storage of cryopreserved embryos? Patients' decisions in view of their child wish.

OBJECTIVE Cryopreservation of supernumerary embryos resulting from IVF treatment offers extra chances to conceive. The objective of this study is to describe patients' decisions to continue or discontinue storage of their embryos after a minimum storage period of 2 years. METHODS Female patients who had embryos stored at the Infertility Centre of the Ghent University Hospital (Belgium) were sent a mail questionnaire to be completed anonymously. RESULTS The questionnaire had a response rate of 79% (326/412). After an embryo storage period of at least 2 years, 40% of the couples who were still together wished to continue storage of their embryos. Half of these had no concrete plans for a transfer and wanted to postpone the decision or keep all options open. For those who decided to discontinue storage (60%), the main reason was the completion of their families. Despite the fact that the patients' child wish was the main factor in their storage decision, two groups of patients with distinct profiles made decisions that were inconsistent with their child wish: those who wanted to continue storage while not wanting a(nother) child (7% of those with no child wish), and those who wanted a(nother) child but decided to discontinue storage (25% of those with a child wish). Overall, these patients more often expressed emotional difficulties regarding this decision. CONCLUSIONS This study demonstrates the importance of gaining more insight into patients' embryo storage decisions (along with their embryo disposition decisions) and into the emotional factors playing a role in patients' decision-making.

Patients' conceptualization of cryopreserved embryos used in their fertility treatment.

BACKGROUND: Although IVF treatments using cryopreserved embryos are offered by most infertility centres, little is known about patients' opinions regarding treatment using these embryos. The objective of this study is to describe how patients think about (treatment with) cryopreserved embryos. METHODS: In-depth interviews based on the method of interpretative phenomenological analysis were conducted with patients undergoing IVF/ICSI treatment at the Department of Reproductive Medicine of the Ghent University Hospital between May and July 2006. Seven couples (one couple considered as one participant) and 11 female patients who attended the clinic without their partner were interviewed. RESULTS: Most participants knew little about medical-technical procedures involved in treatment with cryopreserved embryos. This was compensated by a high confidence in the medical team. However, seven of the eighteen participants thought the quality of cryopreserved embryos diminished during the storage period. A lack of knowledge about medical-technical procedures was compensated by metaphors related to other domains of experience, especially kitchen metaphors: this might add to the belief that frozen embryos have an expiry date. However, none of the patients who considered treatment with cryopreserved embryos as less effective ever thought of refusing this type of treatment or discussed this with medical staff. In addition, patients rarely discussed the moral status of their cryopreserved embryos. CONCLUSIONS: The beliefs of infertile patients about (the effectiveness of treatment with) cryopreserved embryos encompass misconceptions, and doubts and fears which may influence their decision-making but which are seldom discussed with the medical staff.

Xenotransplantation of cryopreserved human ovarian tissue into murine back muscle.

BACKGROUND: Ovarian tissue (OT) cryopreservation and transplantation are options for fertility preservation in young female cancer patients. METHODS: We investigated xenotransplantation of human OT into back muscle (B) of severe combined immunodeficiency mice. OT follicle content was evaluated by stereomicroscopy and pre-transplantation. Xenograft survival, follicular development (with/without FSH administration), apoptosis and vascularization were compared in B- versus K-site (under the kidney capsule) several times after grafting using histology, immunohistochemistry and magnetic resonance imaging. In vitro maturation (IVM) was also performed. RESULTS: Anastomoses which developed from existing human and invading murine vessels were seen in OT at both sites, but angiogenesis was more prominent at the B- than K-site (P < 0.001). Vascularization and follicle size were correlated in the B-group (Spearman's coefficient 0.73; P < 0.001). FSH increased early (8 days) micro-vessel formation in B but not in K grafts (P < 0.0001, versus no FSH). B-site grafts showed a better histological morphology and survival (P = 0.0084), formation of larger antral follicles (P = 0.005), more metaphase-II (MII) oocytes, growing follicles (P = 0.028) and slightly fewer apoptotic follicles than K grafts. One MI oocyte from B underwent IVM and reached MII stage next day. CONCLUSIONS: To our knowledge, this is the first report of MII and IVM-MII oocytes obtained from B xenografts. We report the largest oval-shaped antral follicles containing an MII oocyte obtained after OT xenotransplantation to date. Xenografting in the mouse B should be further explored as a method for human OT transplantation.

Reflections by patients who undergo IVF on the use of their supernumerary embryos for science.

Interviews were conducted with patients undergoing treatment at the department for reproductive medicine at the University Hospital of Ghent, Belgium to describe how patients think about the use of embryos for science and how patients' views are related to their decision whether or not to donate their supernumerary embryos for science. Most participants knew little about the use of embryos for science. The perception of science and scientists, rather than the perception of the embryo, played an important role in the disposition decision making. The feeling of not having control over what would happen to their embryo and the fear that scientists would allow their embryo to develop into children were the main arguments against donation. This showed the importance of information about scientific research with embryos, such as the 14-day limit to keep embryos alive. Half of the participants pictured the medical team as the requesting party for their embryos and those who were not willing to donate indicated that they could be persuaded when asked directly by their caregivers. In conclusion, this study suggests that the perception of science, rather than the perception of the embryo, plays an important role in the decision to donate for science.

Infertility patients' beliefs about their embryos and their disposition preferences.

BACKGROUND: Little research has been done on how beliefs of infertility patients about their embryos are related to their disposition decisions. The objective of this study was to describe how patients speak about their embryos, in moral (e.g. status) and non-moral terms, and to investigate how patients' narratives are related to their disposition preferences. METHODS: In-depth interviews based on the method of interpretative phenomenological analysis were conducted with patients undergoing IVF/ICSI treatment between May and July 2006. RESULTS: Seven couples and 11 female patients were interviewed. Six major themes emerged from the narratives of the participants when they spoke about their embryos: (i) a medical-technical perspective; (ii) feelings; (iii) genetic link to oneself and/or one's partner; (iv) symbolic meaning of the relationship between the infertile partners; (v) moral status and (vi) instrumental value. All but two participants spontaneously considered the embryo disposition options as a two-stage decision sequence. In the first step, they considered donation to another couple for reproductive purposes. At this stage, the presence of the themes 'genetic link' and 'symbol of the relationship' was linked with a clear reluctance to donate. In the second step of the decision-making process, the option of donation for research and discarding were considered. At this stage, participants' confidence in medical science and the instrumental value they attached to the embryo were related to their decisions. CONCLUSIONS: Patients' conceptualization of their embryos plays an important role in embryo disposition decisions. Our research showed that patients deal with these decisions in a two-stage decision sequence.

Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial.

BACKGROUND: It has been suggested in the literature that low-dose aspirin leads to an increased number of oocytes in IVF/ICSI as well as a higher pregnancy rate. The aim of the present study was to investigate the effect of daily administration of low-dose aspirin, compared with placebo, on pregnancy rate in IVF and ICSI. METHODS: This study was a prospective, randomized, double-blind placebo controlled trial, performed in the fertility centre of the University Hospital of Ghent. Concealed allocation by computerized randomization was done by the central pharmacy of the hospital. Daily oral administration of aspirin 100 mg or placebo started before stimulation and was continued until confirmation of pregnancy by detection of fetal heart activity on ultrasound. The primary outcome measure assessed in this trial was clinical pregnancy rate per cycle. RESULTS: Two hundred and one couples were included in this study, 193 women (aspirin group n = 97, placebo group n = 96) started treatment and 181 underwent an embryo transfer. There were 31 clinical pregnancies (31/97 or 32%) in the aspirin group versus 30 (30/96 or 31%; P = 0.916; OR 1.033; 95% CI 0.565-1.890) in the placebo group. CONCLUSIONS: This randomized controlled trial could not show a significant difference in clinical pregnancy rate between the aspirin and the placebo group in a first or second IVF/ICSI cycle. Given the lack of evidence for a beneficial effect of low-dose aspirin, it appears that low-dose aspirin should not be prescribed routinely in IVF/ICSI treatment. ClinicalTrials.gov Identifier: NCT00644085.

Cross-border reproductive care in Belgium.

BACKGROUND: Cross-border reproductive care indicates the cross-border movements made by patients to obtain infertility treatment they cannot obtain at home. The problem at present is that empirical data on the extent of the phenomenon are lacking. This article presents the data on infertility patients going to Belgium for treatment. METHODS: A survey was conducted among the centres for reproductive medicine that are allowed to handle oocytes and create embryos (B-centres). Data were collected on the nationality of patients and the type of treatment for which they attended during the period 2000-2007. RESULTS: Sixteen of 18 centres responded to the questionnaire. The flow of foreign patients has stabilized since 2006 at approximately 2100 patients per year. The majority of foreign nationals seeking treatment in Belgium were French women for sperm donation. The next highest group was patients entering the country to obtain ICSI with ejaculated sperm. CONCLUSIONS: There are clear indications that numerous movements are motivated by the wish to evade legal restrictions in one's home country, either because the technology is prohibited or because the patients have characteristics, which exclude them from treatment in their own countries.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Back muscle as a promising site for ovarian tissue transplantation, an animal model.

BACKGROUND: The aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments in murine hosts. We compared the transplantation to the back muscle (B) versus the kidney capsule (K) in a mouse allograft model. METHODS: Hemi-ovaries from 12-day-old mice were allografted into B and K of bilaterally ovariectomized same strain recipients which had undergone gonadotrophin stimulation (n = 15). Graft survival after 27 days, angiogenesis and follicle development were scored and compared to age-matched control ovaries (38-day old, n = 5). The ability of oocytes to be fertilized was studied after IVF, ICSI and embryos were transferred to recipient mothers. Anti-mouse CD 31+ antibody was used to evaluate neo-vascularization in grafts. RESULTS: Primordial follicle survival was higher (P < 0.01) and vascular support was better (P < 0.01) in B- than in K-grafts. From 34 oocytes retrieved from B-grafts (15 metaphase I, of which 14 matured in vitro, and 19 collected at metaphase II), 18 morulae were obtained. Transfer of 12 embryos obtained by ICSI led to three live offspring, and transfer of six IVF embryos to another recipient mother yielded four offspring, one of which was born dead and one showed placental anomalies. CONCLUSIONS: The back muscle is a promising site for ovarian allografts in mice. This is the first report of live offspring obtained after back muscle grafting using both IVF and ICSI.

PCOS in lesbian and heterosexual women treated with artificial donor insemination.

It has been claimed that the prevalence of polycystic ovary syndrome (PCOS) is significantly higher in lesbian compared with heterosexual women. The present study tried to corroborate this finding in a population of lesbian and heterosexual women consulting for artificial insemination with donor spermatozoa (AID) in the authors' infertility centre. Separate Rotterdam criteria were compared, as well as the outcome of AID. Data were collected from patient files and 174 lesbian and 200 heterosexual women were included in this study. The diagnosis of PCOS was made following the Rotterdam PCOS consensus workshop group. A total of 8.0% of the lesbian women had PCOS compared with 8.7% of the heterosexual women. Concerning the presence of polycystic ovaries and cycle length and regularity, no significant differences were found. Conclusions about hirsutism and chemical hyperandrogenism were not made. Statistical analysis did not show any difference for the type and outcome of treatment. This study does not confirm a link between sexual orientation and the diagnosis of PCOS. The absence of a significant difference in therapy type and outcome emphasizes that there is no difference in (in)fertility rates between the study groups.

Delayed structural development in an acardiac fetus: an echographic observation.

INTRODUCTION: Twin reversed arterial perfusion (TRAP) is a rare condition in monozygotic multiple gestations, caused by the formation of large arterio-arterial anastomoses. Only few serial ultrasound observations are reported, showing the delayed development in the recipient twin. CASE: A monochorionic monoamniotic twin pregnancy with discordant growth was diagnosed at 11 weeks and 4 days in a 28-year-old primigravida. The smaller twin, who first appeared as an amorphic mass, showed growth and developed a clearly recognizable spine and lower limbs in further ultrasound examinations. CONCLUSION: This case illustrates that developmental arrest in acardiac fetuses does not affect the total organism at one time, but that some embryonic fields keep on developing into macroscopically recognizable structures.

Management of patients with non-atypical and atypical endometrial hyperplasia with a levonorgestrel-releasing intrauterine system: long-term follow-up.

OBJECTIVES: Levonorgestrel (LNG), delivered locally into the uterine cavity has a profound effect on the endometrium. The aim of the study was to use a LNG intrauterine system to treat non-atypical and atypical endometrial hyperplasia in women and to evaluate the long-term cure (remission) rate. METHODS: Each of the 20 women in the study, of whom eight were diagnosed with atypical hyperplasia, received a LNG-IUS, releasing 20 microg LNG/day. The study is a non-comparative study with long-term follow-up (range 14-90 months). RESULTS: All women developed a normal endometrium, except one asymptomatic woman with atypical hyperplasia who still had focal residual non-atypical hyperplasia at 3 years follow-up in the presence of a thin (< 4 mm) endometrium. CONCLUSION: Continuous intrauterine delivery of LNG appears to be a promising alternative to hysterectomy for the treatment of endometrial hyperplasia and could enhance the success rate when compared with other routes of progestagen administration as well as intrauterine progesterone delivery. The significant reduction of the PR expression observed during treatment with the LNG-IUS appears to be a marker for the strong antiproliferative effect of the hormone at a cellular level resulting in an inhibition of estrogen bioactivity and endometrial suppression.

Vulvovaginal candidiasis in a Flemish patient population.

Increased resistance to fluconazole has been reported in oral, oesophageal and urinary Candida isolates, but this has not been observed commonly in genital tract isolates. The rate of isolation of Candida spp. and their susceptibility to amphotericin B, flucytosine and azoles were determined in a number of clinical practices in the city of Ghent, Belgium. Patients with symptomatic vulvovaginal candidiasis (VVC) were treated with fluconazole, and the mycological and clinical outcomes were evaluated. Isolates were identified as Candida albicans (78.6%), Candida guilliermondii (17.3%), Candida glabrata (2.6%) and Candida dubliniensis (1.3%). The rates of mycological and clinical cures were 79.5% and 100%, respectively. Women with recurrent VVC were infected more frequently by non-albicans Candida spp., but no association was found between the use of antifungal agents and the presence of non-albicans spp. In-vitro resistance to fluconazole was not detected, even among subsequent Candida isolates from nine patients for whom mycological cure was not achieved.

[Cytogenetic analysis of spermatozoa in a patient with chromosome constitution 45,X/46,X,r(Y) by intracytoplasmic injection into mouse oocytes].

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 46,X,r(Y)/45,X and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 46,X,r(Y)/45,X patient and in the control group did not differ from 1:1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.

[Cytogenetic analysis of human spermatozoa using intracytoplasmic sperm injection into mouse oocytes].

The chromosome complement of human spermatozoa has been analyzed after their intracytoplasmic injection into unfertilized mouse oocytes. A total of 427 metaphase plates have been obtained, including 176 metaphase plates from spermatozoa with normal head morphology (108 and 68 spermatozoa from patients with normal (the control group) and abnormal spermogram parameters, respectively), and 251 metaphase plates from spermatozoa with abnormal heads (76, 91, 67, and 17 spermatozoa with large, amorphous, elongated, and round heads, respectively). The frequency of chromosome abnormalities in the control group is 26.1%, with hyperploidy, hypoploidy, and structural aberrations accounting for 7.4, 12.3, and 6.4% of the abnormalities, respectively. In none of the groups did the ratio between the numbers of X- and Y-bearing spermatozoa significantly differ from 1 : 1. The diploidy frequency was significantly higher in spermatozoa with large and amorphous heads compared to the control group (2.36, 3.29, and 0%, respectively). None of the groups of spermatozoa differed from the control group with respect to the frequency of structural aberrations. The type of the abnormal head morphology has been found to be correlated with the sperm chromosome complement.

Induction of multiple follicular development by a single dose of long-acting recombinant follicle-Stimulating hormone (FSH-CTP, corifollitropin alfa) for controlled ovarian stimulation before in vitro fertilization.

In a first feasibility study, the efficacy and safety of a single dose of recombinant long-acting FSH (FSH-CTP) were investigated in in vitro fertilization (IVF) patients undergoing controlled ovarian stimulation with a flexible GnRH antagonist protocol. Eligible subjects were randomized to receive a single dose of 120 micro g (n = 25), 180 microg (n = 24), or 240 microg (n = 25) corifollitropin alfa (FSH-CTP) or to start daily fixed doses of 150 IU recombinant FSH (rFSH) (n = 24, reference). Subjects who received a single dose of FSH-CTP continued 1 wk after injection (treatment d 8) with fixed daily doses of 150 IU rFSH (Puregon/Follistim) until the day of triggering final oocyte maturation. The terminal half-life of FSH-CTP was, on average, 65 h and dose independent. Cycle cancellation before human chorionic gonadotropin (hCG) administration occurred in only three subjects treated with FSH-CTP. The median duration of stimulation was 10.0 d in each FSH-CTP group and 9.0 d in the daily rFSH group. The total number of follicles at least 11 mm at stimulation d 8 and at the day of hCG administration tended to increase with dose of FSH-CTP, although a significant dose-response relationship was revealed only for the number of follicles at least 15 mm on the day of hCG (P = 0.03). Serum estradiol levels and inhibin-B levels were not significantly different between the four groups on d 8 and on the day of hCG. In total, 12 subjects (17.6%) in the FSH-CTP groups and two subjects (8.3%) in the rFSH group experienced a premature LH rise (defined as LH >or= 10 IU/liter) before the start of the GnRH antagonist (P value not significant between groups). This relatively high incidence of women demonstrating an early LH rise in the FSH-CTP groups may be related to the higher initial rises of serum estradiol and the use of a flexible GnRH antagonist protocol. The mean number of oocytes recovered per started cycle was higher in FSH-CTP-treated subjects compared with rFSH-treated subjects (significant at P = 0.03 for the 240- microg FSH-CTP group), but no difference could be noted between the number of good quality embryos (range of means, 3.8-4.8 per attempt), and equal numbers of embryos were available for embryo transfer. In summary, FSH-CTP appeared to be a potent inducer of multiple follicular growth; additional research will be needed to select the optimal FSH-CTP dose and treatment time interval.

Steroid determinations in human ovarian follicular fluid using reversed phase liquid chromatography.

A method is presented, based on high performance liquid chromatography (HPLC) with u.v. absorbance detection, to simultaneously analyse all major unconjugated steroids in ovarian follicular fluids. The total analysis time is only 30 min. The use of a 3 mm i.d. column allows us to obtain detection limits for 3-oxo-4-ene steroids of 2 ng/ml. Calibration curves are linear in the 10-20,000 ng range per injection. Excellent agreement is obtained with the results using a previously published gaschromatography method.

Follicular fluid steroid levels in relation to oocyte maturity and in vitro fertilization.

Steroid levels in follicular fluid (FF) obtained from stimulated ovaries in patients undergoing in vitro fertilization (IVF) were measured by capillary gas chromatography. The correlation between these levels and the maturity of the oocyte, judged from the morphology of the oocyte corona cumulus complex (OCCC) and the fertilizability of the oocytes was analysed. Oocyte maturity was associated with higher FF levels of progesterone, 17-hydroxyprogesterone, 16 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone. Follicular fluids containing oocytes that became fertilized had significantly higher levels of 20 alpha-dihydroprogesterone and progesterone and lower levels of androstenedione. Of all the steroids determined, 20 alpha-dihydroprogesterone provides the most significant group differences. Enhanced 20 alpha-dihydrogenation in the presence of decreased 16 alpha- and 17-hydroxylation appears to be an important characteristic of the ultimate ripening stages and early luteinization, at least in stimulated cycles.