Different patterns of structural luteolysis in the human corpus luteum of menstruation

Structural luteolysis is a complex process responsible for the elimination of the corpus luteum (CL). The aim of this study was to analyse the luteolytic process of the CL of menstruation. For this, we have morphologically studied 654 ovaries from 340 cycling women. Apoptotic cells were observed almost exclusively during the perimenstrual period and were extremely scarce at advanced stages of involution. Steroidogenic luteal cells surviving to the perimenstrual apoptotic wave underwent characteristic degenerative changes, consisting of intense cytoplasmic vacuolation, expression of macrophage markers and accumulation of lipofuscin pigment, and they persisted for long periods of time. Accumulation of corpora albicantia (CA) was observed in only 25% of a subset of 168 women, whereas 28% showed involuting CL without hyalinization, consisting of clusters of pigment-filled cells, and 46.4% showed ovaries with a mixture of CA and involuting CL without hyalinization or involuting CL with intermediate features. Evolution of the CL towards CA seemed to be related to the presence of a large, blood-filled cavity. The data from this study suggested that different patterns of structural luteolysis exist during CL involution, and that the final fate of the involuting CL is dependent on the presence of a large, central, blood-filled cavity.     

Regulation of corpus luteum function

The effects have been studied of different ovulation inductionregimens [either domiphene citrate or buserelin in combinationwith human menopausal gonadotrophin (HMG)] on the circulatingconcentrations of progesterone, oestradiol, relaxin and humanchorionic gonadotrophin (HCG) during the first trimester ofpregnancy. Ovulation induction with clomiphene resulted in elevatedconcentrations of gonadotrophins in both phases of the cycle,while during ovulation induction with buserelin, gonadotrophinconcentrations were elevated in the follicular phase only. Theconcentrations of all corpus luteum products were greater inclomiphene pregnancies compared with spontaneous pregnancies,but only oestradiol and relaxin concentrations were greaterin clomiphene pregnancies compared with buserelin pregnancies.The concentrations of HCG were significantly reduced in clomiphenepregnancies compared to natural pregnancies. Relaxin concentrationswere significantly higher from 7 weeks gestation in buserelincompared with spontaneous pregnancies, while progesterone, oestradioland HCG concentrations were not consistently different. Consistentassociations were found between relaxin and HCG concentrationsin spontaneous pregnancies and between the concentrations ofrelaxin and both progesterone and oestradiol in pregnanciesachieved after ovulation induction. These data suggest that(i) given the similarity in the circulating concentrations ofHCG, the relatively lower circulating gonadotrophin concentrationsduring the luteal phase of the cycle of conception result inreduced circulating concentrations of oestradiol and relaxin;while in the case of relaxin this effect is partially reversible,there is no evidence that this is so for oestradiol; (ii) synthesisof progesterone in the corpus luteum is less affected by lowerconcentrations of gonadotrophins during the luteal phase; (iii)ovulation induction with clomiphene results in pregnancies withlower concentrations of HCG, suggesting that trophoblast functionmay be impaired; and (iv) corpus luteum function is linked withplacental steroidogenesis.     

Discovery of LH-regulated genes in the primate corpus luteum

Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n = 3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n = 3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11,600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT-PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n = 3/stage) between the mid (day 6-8), late (day 14-16), or very late (day 18-19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited > or = 2-fold change in expression after ANT. Of the 25 cDNA exhibiting a > or = 6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT-PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P < 0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle.     

Salivary progesterone measurements reveal corpus luteum defects which respond poorly to hormonal treatments in operated mild and moderate endometriosis.

Thirty-five women who had been operated on for mild or moderate endometriosis were evaluated through 40 menstrual cycles by daily measurements of salivary progesterone concentrations. As controls, 17 women with normal ovulatory cycles and no endometriosis were studied. In the endometriosis group, 22 cycles were unstimulated, seven were stimulated by clomiphene citrate and 11 with clomiphene citrate, human menopausal gonadotrophin and human chorionic gonadotrophin. A variety of aberrations in profiles of salivary progesterone secretion was detected in all the groups of endometriosis patients and the frequency of normal cycles was significantly lower than in controls (14-18% versus 82%, P less than 0.01). The response to the treatments varied greatly within the groups. In conclusion, the present data demonstrate that patients with endometriosis have a variety of defects in the menstrual pattern of salivary progesterone secretion and that their corpus luteum function responds poorly to stimulatory treatments.     

Distribution of leukocyte subpopulations in the human corpus luteum.

Cytokines, as secreted products of leukocytes, have roles in many organs of the body via paracrine or autocrine mechanisms. In the present study, we demonstrate by immunocytochemistry the leukocytes present in the human corpus luteum in order to investigate further the relationship between leukocytes, cytokines and corpus luteum function. Ten intact corpora lutea were collected from female patients who had no apparent ovarian disease. The mean age of these patients was 37 years (range 23-55 years). Frozen and paraffin sections were subjected to analysis using monoclonal antibodies which were specific to leukocyte marker antigens. The results showed that there are macrophages, cells positive for leukocyte common antigen (LCA), T lymphocytes including T helper/inducer (T4) cells, T cytotoxic/suppressor (T8) cells and activated T (Ta) cells (interleukin-2 receptor-positive cells), monocytes and natural killer (NK) cells but not B lymphocytes present in the human corpus luteum. The distribution of the leukocytes present in the different parts of the corpus luteum was found to be in the order: theca-luteal area greater than loose connective tissue area greater than granulosa-luteal area. Macrophages and T lymphocyte subsets comprised the main components of the total leukocytes in the human corpus luteum. Ta cells were only localized in the loose connective tissue of the corpus luteum. In most cases, macrophages, LCA cells and T4 cells tended to be situated in a single cell layer on the edge of the theca-luteal area and surrounding the granulosa-luteal area. These results suggest that the leukocytes may act to a greater extent in the theca-luteal area than in the granulosa-luteal area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the corpus luteum in the ovulatory polycystic ovary.

BACKGROUND: Women with polycystic ovaries (PCO) have a wide spectrum of presentation from anovulation and amenorrhoea to apparently regular, ovulatory menstrual cycles. We have recently reported a subtle defect in steroidogenic function in the luteal phase in the latter and an increase in the number of degenerating corpora lutea (CL) were observed in ovulatory PCO (ovPCO) during dissection. The possibility was therefore investigated of differences in structure or degeneration in CL formed during ovulatory cycles in women with PCO. METHODS: This study compared the histology of the CL in ovPCO with that in the normal ovary. Corpora lutea were collected from nine normal ovaries (days 1-27 of the cycle) and from 13 women with ovPCO (days 5-38). RESULTS: Variations in the degree of regression, both in relation to onset of menses and between different areas within individual CL, were recorded in both groups. During development and regression no obvious differences were observed between either group apart from an apparent increase in luteal haemorrhage, which was more common and more extensive in CL from PCO. CONCLUSIONS: The findings suggest that possible luteal phase abnormalities of steroid secretion in women with ovulatory PCO are not associated with obvious morphological defects in the CL, however it is possible that the persistence of luteal structures seen in PCO was a consequence of increased luteal haemorrhage.     

Localization of prostaglandin endoperoxide synthase in the human corpus luteum

Prostaglandins have been implicated in both maintenance and luteolysis of the primate corpus luteum. Central to the production of prostaglandins is the enzyme prostaglandin endoperoxide synthase (PGHS). In the present study, we identified the cell types which contain PGHS in 44 human corpora lutea, using immunoperoxidase staining techniques. Intense granular staining was present in the cytoplasm of granulosa lutein cells of tissues obtained from the mid-luteal phase. Theca lutein cells demonstrated a diffuse cytoplasmic staining which was less intense than that observed in granulosa lutein cells. Staining appeared less intense in tissues from the early or late phase. Ovarian stromal cells demonstrated little or no PGHS immunoreactivity. PGHS staining in the corpus luteum of pregnancy was similar in intensity and cell distribution to that of mid-luteal corpus luteum. In summary, human corpus luteum contains immunoreactive PGHS which localized mainly to well-differentiated granulosa lutein cells.     

Extracellular matrix of the human cyclic corpus luteum

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV alpha1 and laminins alpha5, beta2 and gamma1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV alpha1 and laminins alpha2, alpha5, beta1 and beta2 localized in the interstitial matrix. Laminin alpha4 and beta1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin alpha1 and alpha3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.     

Cell proliferation and vascular morphology in the marmoset corpus luteum

Luteal formation is associated with angiogenesis and low progesterone production. Maximal mid-luteal phase progesterone production is concurrent with extensive vascularization, and luteolysis occurs when steroidogenesis decreases. Angiogenic cell proliferation and vascular changes have not been examined in the marmoset. The aim of this study was to examine vascular morphology throughout the luteal phase by identifying: (i) von Willebrand factor VIII antigen (vW)-immunopositive endothelial cells; (ii) Ki67-positive proliferating cells; and (iii) bromodeoxyuridine-positive proliferating cells. Marmoset corpora lutea were examined throughout the cycle, and natural regression was compared with induced luteolysis after administration of a prostaglandin F(2alpha) analogue or gonadotrophin-releasing hormone (GnRH) antagonist. Steroidogenic and endothelial cells were positive for proliferation markers. Endothelial cell proliferation was highest during luteal formation, then decreased and remained low during the luteal phase and functional regression, however endothelial cell proliferation increased during structural regression. Endothelial cell proliferation was unchanged by induced regression. The area of vW immunostaining was highest during luteal formation, decreased thereafter and remained constant during the luteal phase and regression. Distribution of immunostaining indicated the presence of an extensive capillary network, but during structural regression the numbers of capillaries decreased and numbers of microvessels increased. These results suggest that vascular changes are concurrent with changes in the functional status of the marmoset corpus luteum.     

Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function

The role of leukocytes and cytokines in ovarian physiology isnow established, although the function of each cell type andcytokine remains to be determined in detail. Current knowledgeof these effects on follicle development, ovulation, luteinizationand luteotrophic process and luteolysis is reviewed. It is possiblethat further research will help to unravel some of the clinicalmysteries in ovarian function, including polycystic ovary syndrome,premature menopause, ovulatory disorders, and luteal phase defect.Furthermore, the increasing use of cytokines and their antagonistsin clinical practice may have significant effects upon reproductivefunction.     

EGF-like growth factors as LH mediators in the human corpus luteum

BACKGROUND: This study aims to investigate the role of epidermal growthfactor-like ligands, amphiregulin (Ar) and epiregulin (Ep),in regulation of apoptosis in luteinized human granulosa cells. METHODS: Luteinized human granulosa cells were obtained from women undergoingIVF treatment. Ar and Ep mRNA levels were measured by real-timeRT–PCR. The rate of apoptosis was measured by TUNEL. Progesteronelevels were measured using radioimmunoassay. Ar- and Ep-inducedactivation of signaling cascades and Ar protein levels weredetected by western blotting. RESULTS: LH stimulation of luteinized human granulosa cells induced biosynthesisof Ar and Ep mRNA in a time-dependent manner. The blockade ofMEK (by U0126) reduced the expression of LH-induced Ar and Epbiosynthesis. Incubation of the cells with Ar and Ep completelyabolished the increase in apoptosis rate induced by serum starvation,and concomitantly caused a pronounced increase in progesteroneproduction. Stimulation of the cells with Ar and Ep also activatedthe ERK and AKT signaling cascades. Finally, we demonstratedthat the pro-survival effect of Ar and Ep is partially dependenton their ability to induce progesterone production. CONCLUSIONS: Ar and Ep serve as pro-survival LH mediators in the human corpusluteum.     

Pregnancy: Corpus luteum failure in ectopic pregnancy

The endocrinology of ectopic pregnancy was studied in orderto investigate the origin of the discordance in the circulatingamounts of human chorionic gonadotrophin (HCG) and those ofoestradiol and progesterone. Serial maternal blood samples wereobtained at 4–9 weeks gestation from 93 patients who becamepregnant following in-vitro fertilization and embryo transferincluding 10 ectopic, 21 anembryonic and 62 normal singletonpregnancies. The samples were analysed for HCG, Schwangerschaftprotein-1 (SP-1), pregnancy-associated plasma protein-A (PAPP-A),progesterone and oestradiol. In ectopic pregnancies, concentrationsof all substances analysed were significantly reduced comparedto singleton pregnancies from 5 weeks gestation (P < 0.05–0.001)but they were not significantly different from those of anembryonicpregnancies. In ectopic pregnancies, associations were foundbetween the concentration of both HCG and SP-1 and those ofprogesterone and oestradiol. No associations were found betweenPAPP-A and any other substances analysed. This may be due toinsensitivity of the PAPP-A assay; alternatively PAPP-A concentrationsmay be differentially reduced in ectopic pregnancy. These findingssuggest that progesterone and oestradiol are derived from thecorpus luteum in early ectopic pregnancy but that the corpusluteum fails rapidly and the dominant source of both hormonesbecomes the trophoblast as early as 5 weeks.     

Expression and regulation of Sprouty-2 in the granulosa-lutein cells of the corpus luteum

Growth factor signalling has important modulatory roles in the process of human follicular growth, oocyte maturation and corpus luteum (CL) formation. Recently, Sprouty-2, an inhibitor of receptor tyrosine kinase (RTK) signalling pathway was advocated as a marker of oocyte competence in the bovine ovary. We sought to study Sprouty-2 expression and regulation in the human ovary. RT-PCR was used to detect Sprouty-2 mRNA in human granulosa-lutein cells (GLC) collected from follicular aspiration of IVF patients. The effect of epidermal and fibroblast growth factors (EGF and FGF) on Sprouty-2 mRNA expression in GLC was studied using quantitative real-time PCR. Immunohistochemistry was performed on cultured GLC, human CL and stimulated rat ovary sections. Sprouty-2 mRNA was expressed in human GLC. EGF and basic FGF, but not FGF4 and FGF10, increased Sprouty-2 mRNA expression in GLC. The Sprouty protein was localized to GLC of early and late human CL but not to the theca cell layer. Immunostaining of developing rat CL confirmed the temporal and spatial expression of Sprouty in humans. The detection of Sprouty-2 mRNA and protein in human GLC may suggest a role for Sprouty-2 during the final stages of follicle maturation and CL formation.     

Recovery of corpus luteum function after prolonged deprivation from gonadotrophin stimulation

Three women with hypogonadotrophic hypogonadism, all desiringpregnancy, participated in a prospective open study attemptingto assess the ability of the human corpus luteum to recoverafter 7 days of deprivation from gonadotrophin stimulation.Follicular growth was induced by gonadotrophins. An endogenousluteinizing hormone (LH) surge was induced by the s.c. injectionof a gonadotrophin-releasing hormone agonist For luteal support,10 mg/day oral medroxyprogesterone acetate were given for 7days, after which a single i.m. injection of human chorionicgonadotrophin (HCG) was administered. Monitoring during thefollicular phase consisted of daily measurements of serum oestradiol,LH and follicle stimulating hormone (FSH) concentrations, andof follicular growth by trans-vaginal ultrasonography. Duringthe luteal phase, monitoring consisted of measurements of serumconcentrations of LH, FSH, oestradiol, progesterone, 17-hydroxy-progesteroneand -HCG. Ovulation and luteinization occurred in two patients,demonstrated by transient marked increases in serum progesteroneand 117-hydroxy-progesterone concentrations which decreasedto basal pre-ovulatory values and increased again followingthe administration of HCG 7 days later. In the third patient,ovulation and luteinization did not occur, and the subsequentadministration of HCG did not result in an increase in progesteroneconcentration. Of the two patients who ovulated, one conceivedand the second had a luteal phase of 15 days duration. Our preliminaryresults suggest that the human corpus luteum can be ’rescued‘and can function normally after 7 days of deprivation from gonadotrophinstimulation in patients with hypogonadotrophic hypogonadism.     

Power and colour Doppler ultrasonography for the evaluation of the vasculature of the human corpus luteum

We used power Doppler imaging to examine neovascularizationin the corpus luteum (CL) in 12 healthy volunteers. We alsoinvestigated whether CL blood flow reflected luteal function.The ratio of the area of vessels in the CL to the area of asectional plane at the maximum diameter of the CL observed bypower Doppler (FA ratio) was used as a quantitative index ofthe vascularity of the CL. The pulsatility index (PI) was significantlylower in ovarian arteries with CL than without CL (P <0.05).Changes in ovarian arterial and intra-luteal PI appeared toreflect physiological changes in the vasculature of the CL.There was no correlation between the volume of the CL or theFA ratio and the concentration of progesterone. The patternof changes in the product of the FA ratio and the CL volumeand in the progesterone concentration was similar. The progesteroneconcentration was positively correlated with this product (r= 0.74, P < 0.01). The product of the FA ratio and the CLvolume plateaued during the mid- to late luteal phase, suggestingthe presence of functional and structural luteolysis. Thesefindings suggest that colour Doppler ultrasonography, includingpower Doppler imaging, can detect physiological changes in theblood flow of the ovary in the luteal phase, and may be a usefulnon-invasive tool for evaluating CL function.     

The endometrium in natural and artificial luteal phases

The normal human endometrium reacts precisely and sen–sitivelyto any hormonal stimulation with predictable changes. If thecorpus luteum develops normally after ovulation, the progesteronesecreted induces specific changes in endometrial glandular andstromal cells that can be dated by dally fine–structuralalterations. With corpus luteum deficiency the endometrial differentationis delayed and remains incomplete. The defect in the differentiationof glands and stroma varies in their distribution and intensityand may be dissociated or coordinated depending upon the causeof the corpus luteum deficiency. The administration of naturalprogesterone in the second half of the cycle prolongs the lutealphase and may result in hypersecretion of glands. In contrast,therapy with synthetic gestagens depresses glandular secretion,induces glandular atrophy and decidualization of the stroma.The various synthetic gestagens differ both quantitatively andqualitatively in their action; depending upon the dosage giventhe endometrium remains in abortive or arrested secretion, domlphenedepresses normal secretion by its antioestrogenk effect whichcauses deficient oestrogen priming. On the other hand, clomiphenecounteracts excessive oestrogen and will normalize the secretionin a deficient luteal phase that was preceded by follicularpersistency. In the artificial cycle, depending upon the stateof endogenous hormonal stimulation, the patients will benefiteither from clomiphene or gonadotrophin to maintain or normalizetheir secretory endometrium.     

Dynamic expression of epoxyeicosatrienoic acid synthesizing and metabolizing enzymes in the primate corpus luteum

Epoxyeicosatrienoic acids (EpETrEs), produced from arachidonic acid via cytochrome P450 (CYP) epoxygenases, regulate inflammation, angiogenesis, cellular proliferation, ion transport and steroidogenesis. EpETrE actions are regulated through their metabolism to diols (dihydroxyeicosatrienoic acids; DiHETrE) via the enzyme soluble epoxide hydrolase (EPHX2). We set out to determine, therefore, whether EpETrE generating (epoxygenases CYP2C8, 2C9, 2C19, 2J2, 1A2 and 3A4) and metabolizing (EPHX2) enzymes are expressed in the primate corpus luteum (CL). CL were isolated from rhesus macaques during the early (day 3-5 post-LH surge), mid (day 6-8), mid-late (day 10-12), late (day 14-16) and very-late (day 17-19: menses) luteal phase of natural menstrual cycles. EPHX2 mRNA levels peaked in mid-late CL (5-fold when compared with early CL, P<0.05) and remained elevated in the late CL. Ablation of pituitary LH secretion and luteal steroid synthesis significantly reduced (P<0.05) EPHX2 mRNA levels in the mid-late CL, with progestin replacement being insufficient to restore its level of expression to control values. EPHX2 protein was localized to large and small luteal cells, as well as vascular endothelial cells. The EpETrE-generating CYP epoxygenase 2J2, 2C9 and 3A4 genes were also expressed in the macaque CL. While CYP2J2 mRNA levels did not significantly change through the luteal phase, CYP2C9 and CYP3A4 levels were significantly (P<0.05) higher in the mid-late phase when compared with the early phase. CYP2C9, 2J2 and 3A4 proteins were each localized to the large luteal cells, with 2C9 and 2J2 also being present in the small luteal, stromal and endothelial cells. These studies demonstrate for the first time that an EpETrE generating and metabolizing system exists in the primate CL, with the latter being regulated by LH and steroid hormone(s).     

Preliminary results on the role of embryonic human chorionic gonadotrophin in corpus luteum rescue during early pregnancy and the relationship to abortion and ectopic pregnancy.

The precise mechanisms by which corpus luteum (CL) function is modulated during early pregnancy are not known. Evidence in failed pregnancies (ectopic, abortions), shows that factors other than human chorionic gonadotrophin (HCG) could be involved in its regulation. The objective of this study was to investigate the dynamics of beta-HCG, progesterone and oestradiol production in early pregnancy and its relation to embryonic quality and topographic localization. Plasma concentrations of progesterone, oestradiol and beta-HCG were studied between days +12 and +21 after an in-vitro fertilization (IVF) embryo transfer in 11 intrauterine pregnancies, 10 intrauterine abortions and seven tubal pregnancies. Tubal pregnancies and abortions were grouped according to doubling time (DT) of HCG. Results showed that oestradiol concentrations were apparently reduced in both ectopic pregnancies and abortions compared with normal pregnancies. The fall in oestradiol concentrations was seen in ectopic pregnancies with an abnormal DT for HCG and in all abortions. When the ectopic pregnancy had a normal DT, oestradiol and progesterone concentrations were normal. In abortions, the fall in oestradiol and progesterone concentrations was less influenced by the DT of HCG. These findings suggest that corpus luteum function depends on an adequate DT of HCG more than an absolute value, and with normal trophoblastic tissue the site of implantation does not affect CL function.     

Impaired corpus luteum function and other undesired results of pregnancies associated with inadvertent administration of a long-acting agonist of gonadotrophin-releasing hormone.

Spontaneous pregnancies associated with inadvertent periconceptional administration of long-acting gonadotrophin releasing-hormone agonist (GnRHa), in in-vitro fertilization, occurred in 11 of 161 patients with non-tubal infertility. All these cases exhibited impaired function of the corpus luteum in terms of declining progesterone levels, despite rising levels of human chorionic gonadotrophin. The patients were categorized according to the timing of GnRHa administration: periovulatory (three cases), midluteal (five cases) and late luteal (three cases). Altogether, of the 11 pregnancies, seven ended with a normal livebirth, three with a preclinical gestation and one with a blighted ovum. It appears that spontaneous pregnancies associated with inadvertent administration of GnRHa are not rare. Awareness for early diagnosis and close hormonal monitoring are recommended for the assessment of corpus luteum function and adequate supplementation.