Selective vitrification of euploid oocytes markedly improves survival, fertilization and pregnancy-generating potential.

Enthusiasm for oocyte cryopreservation has been limited by poor pregnancy rates per thawed metaphase II (MII) oocytes (<4%) and low implantation rates per embryos. The reasons relate to technical limitations in the freezing process, and the fact that <40% of oocytes are euploid and unable to produce 'competent' embryos. Comparative genomic hybridization was performed on the first polar body (PB-1) of 323 MII oocytes retrieved from 16 donors. Of these, 111 were euploid, and were vitrified. Seventy-five of 78 vitrified oocytes (96%) survived warming and were fertilized using intracytoplasmic sperm injection. Thirty-one (41%) subsequently developed into expanded blastocysts, of which no more than two were subsequently transferred per uterus to 16 out of 19 prospective embryo recipients. Twelve of 19 (63%) recipients produced 17 healthy babies (eight singletons, three twins, and one set of triplets) One twin pregnancy miscarried in the late first trimester The birth rate per transfer of a maximum of two blastocysts to 16 recipients was 75%. The implantation rate per vitrified euploid oocyte was 27%. This study showed a six-fold improvement in pregnancy rate per cryopreserved oocyte over previous reports and a marked improvement in implantation rate. If independently validated, this approach could open the door to commercial egg cryobanking, significantly expanding women's reproductive choices.

Antibodies to phosphatidylethanolamine and phosphatidylserine are associated with increased natural killer cell activity in non-male factor infertility patients.

Antiphospholipid antibodies (APA) have been identified in patients with recurrent pregnancy loss and IVF failure. Of these, antiphosphatidylethanolamine (aPE) and antiphosphatidylserine (aPS) may have special significance. A link between increased natural killer cell activity (NKa+) and trophoblast cell apoptosis has also been reported. This study was undertaken to determine how the APA profile was associated with peripheral NK cell activity. We evaluated 197 female IVF candidates for APA and NKa. Eighty-nine patients (45%) were APA+ and of these, 51 (57%) were aPE/aPS+. Fifty-four patients (27%) had increased NK cell activity. Some 51% of APA+ and 78% of aPE/aPS+ patients had increased NK cell activity compared with 8% and 13% when APA and aPE/aPS tested negative respectively (P: < 0.0001). Non-male factor infertility patients were APA+ and NKa+ in 57% and 34% of cases respectively, compared with 19% and 13% if a pure male factor was present. Some 88% of aPE/aPS+, non-male factor patients had increased NK cell activity, compared with 12% who tested aPE/aPS negative (P: < 0.0001) and 25% of aPE/aPS+, isolated male factor patients (P: < 0.0001). These findings establish a direct relationship between APA (specifically aPE/aPS) and increased peripheral NK cell activity among non-male factor infertility patients. It is possible that APA do not directly cause reproductive failure but rather function as markers or intermediaries for an underlying, abnormal activation of cellular immunity.

The selective use of heparin/aspirin therapy, alone or in combination with intravenous immunoglobulin G, in the management of antiphospholipid antibody-positive women undergoing in vitro fertilization.

PROBLEM: The effect of mini-dose heparin/aspirin (H/A) alone vs. combined intravenous immunoglobulin G (IVIg) and H/A on in vitro fertilization (IVF) birthrates in women who test seropositive for antiphospholipid antibodies (APA+) was evaluated, as was the question of whether outcome is influenced by the gammaglobulin isotype(s) or the phospholipid (PL) epitope(s) to which the APAs are directed. METHOD OF STUDY: A case-control study was conducted in three phases, spanning a 4-year period, in a multicenter clinical research environment. Six hundred eighty-seven APA+ women, who were younger than 40 years and who each, completed up to three consecutive IVF/embryo transfer cycles within a 12-month period, were given either H/A alone or H/A in combination with IVIg. Birthrates relative to the type of immunotherapy (i.e., H/A alone and H/A with IVIg) and APA profile were the main outcome measurements. RESULTS: In phase I, 687 women who tested APA+ to one or more PL epitopes underwent two or fewer IVF attempts for a total of 1050 IVF cycles. Four hundred seventy-seven (46%) births occurred in 923 IVF cycles in which H/A alone was administered. Twenty-two (17%) births occurred after 127 IVF cycles in which H/A was not administered. In phase II, 322 of 687 women tested positive for a single APA subtype. These subjects underwent up to two consecutive IVF attempts for a total of 521 IVF cycles while receiving H/A alone. The birthrate was significantly lower for women whose APAs were directed toward phosphatidylethanolamine (PE) or phosphatidylserine (PS) involving IgG or IgM isotypes than for women who had any other APA (17% vs. 43%). In phase III, 121 women who did not achieve live births after two consecutive IVF attempts in which H/A alone was administered received IVIg in combination with H/A during their third consecutive IVF cycle. The birth rate was 41% after these IVF cycles when anti-PS or anti-PE involving IgG or IgM isotypes were present, as compared with 17% when H/A alone was administered. The IVF outcome did not improve when IVIg was administered in association with any other single APA. CONCLUSIONS: The treatment of APA+ women with H/A alone improves IVF birthrates. This benefit is selective in that it does not apply in cases in which IgG- or IgM-related APAs are directed against PE or PS. In such cases, the addition of IVIg significantly improves the outcome.

A rational basis for the use of combined heparin/aspirin and IVIG immunotherapy in the treatment of recurrent IVF failure associated with antiphospholipid antibodies.

PROBLEMS: 1) Does the administration of heparin and aspirin (H/A) in combination with intravenous immunoglobulin G (IVIG) improve in vitro fertilization (IVF) implantation and birth rates in patients with recurrent IVF failure? 2) Is the effect of such treatment related to the antiphospholipid antibody (APA) status of the patients concerned? METHOD OF STUDY: Subjects consisted of 89 women younger than 36 years of age whose infertility was a result of causes other than male infertility and who had experienced four or more failed IVF/embryo transfer procedures. Fifty-two women were APA+ (group A), and 37 were APA- (group B). All patients, regardless of their APA status, received H/A (5000 U sq bid), aspirin (81 mg po qd) from the inception of menotropin therapy along with IVIG (20 g) through a single infusion 3 to 10 days before egg retrieval. RESULTS: Twenty-two (42%) of group A and 7 (19%) of group B patients achieved live births (P = 0.020). CONCLUSIONS: IVF outcome is significantly improved when H/A and IVIG are administered to APA+ women with repeat IVF failures. APA- women do not seem to benefit from such treatment.

The use of combined heparin/aspirin and immunoglobulin G therapy in the treatment of in vitro fertilization patients with antithyroid antibodies.

PROBLEM: To compare the effect of heparin/aspirin therapy alone vs. heparin/aspirin in combination with intravenous immuno-globulin (IVIg) immunotherapy on in vitro fertilization (IVF) outcome of patients who test positive for antithyroid antibodies (ATAs). METHOD OF STUDY: Eighty-two women younger than 40 years of age whose infertility was related exclusively to female causes were evaluated. All tested positive for organ-specific antithyroid antibodies (antimicrosomal and/or antithyroglobulin antibodies), but negative for antiphospholipid antibodies. Thirty-seven of these women (group A) received H/A alone, whereas 45 (group B) received heparin/aspirin in combination with IVIg. RESULTS: Ten (27%) of women in group A and 23 (51%) of women in group B achieved live births after completion of a single IVF/embryo transfer cycle (P = 0.027). CONCLUSION: We conclude that IVIg therapy significantly improves IVF success rates in ATA+ women.

'Prolonged coasting': an effective method for preventing severe ovarian hyperstimulation syndrome in patients undergoing in-vitro fertilization.

Over a 4 year period ending 1 January 1995, 51 women scheduled for in-vitro fertilization (IVF) and embryo transfer were inadvertently severely overstimulated with menotrophins, as evidenced by the development of > 29 ovarian follicles in association with peak plasma oestradiol concentrations of > 6000 pg/ml. Accordingly, these women were at great risk of developing life-endangering complications associated with severe ovarian hyperstimulation syndrome (OHSS). Treatment involved withholding the administration of both menotrophins and human chorionic gonadotrophin for a number of days, while continuing gonadotrophin-releasing hormone agonist until the plasma oestradiol concentration fell to < 3000 pg/ml ('prolonged coasting'). The mean number of oocytes retrieved was 21.0, while the mean number of embryos transferred per procedure was 5.4. There were 21 clinical pregnancies (i.e. pregnancy rate of 41% per oocyte retrieval), 19 of which resulted in live births (i.e. a live birth rate of 37% per oocyte retrieval). Two pregnancies miscarried and there were four multiple gestations (three sets of twins and one set of triplets). None of the women developed severe OHSS. Prolonged coasting is an effective method of preventing the occurrence of severe OHSS without necessitating the cancellation of the IVF cycle or compromising success rates.

High fecundity rates following in-vitro fertilization and embryo transfer in antiphospholipid antibody seropositive women treated with heparin and aspirin.

This study was undertaken to explore whether intervention with heparin and aspirin (H/A) in selected patients undergoing in-vitro fertilization (IVF) and embryo transfer could improve fecundity rates. Specifically, it explored the possibility that women diagnosed with organic pelvic disease who demonstrated antiphospholipid antibodies (APA) could benefit from H/A administration in a similar manner to that used in patients with recurrent pregnancy loss. We used an enzyme-linked immunosorbent assay for six different phospholipids to identify patients who expressed APA before they underwent IVF/embryo transfer. This study was confined to the first IVF/embryo transfer cycle that followed assessment of APA status and accordingly, the number of IVF/embryo transfer cycles corresponds with the number of patients treated. APA seropositive patients were treated with aspirin, 81 mg orally q.d., and heparin 5000 IU s.c. b.i.d., beginning on day 1 of controlled ovarian stimulation. The endpoint for success was a live birth or an ultrasound confirming fetal cardiac activity (a viable pregnancy). The prevalence of APA in patients diagnosed with organic pelvic disease (53%) was much higher than in those without female pathology (14%). The administration of H/A to APA seropositive patients significantly (P < 0.05) improved the viable pregnancy rate (49%) compared to the untreated APA seropositive group (16%). The viable pregnancy rate for APA seropositive women treated with H/A was also significantly (P < 0.001) higher than for untreated APA seronegative patients (27%). We conclude that all women undergoing IVF/embryo transfer should be tested for APA prior to initiating ovarian stimulation and those with APA seropositivity should be treated with H/A.

Management of suboptimal sonographic endometrial patterns in patients undergoing in-vitro fertilization and embryo transfer.

A total of 816 women who underwent 1332 cycles of ovarian stimulation for in-vitro fertilization and embryo transfer (IVF/ET) had sonographic assessments of the endometrium within 2 days of oocyte retrieval. Endometrial linings were classified on the basis of thickness and echogenicity, using a grading system described previously. Grades I and IIB ('poor') were associated with a 6% viable pregnancy rate (advanced beyond 12 weeks' gestation) compared with a 29% rate for Grade IIA ('optimal'). In a subset of 112 women with poor endometrial linings during natural cycles, eight out of 21 women (38%) under 40 years of age developed optimal linings following ovarian stimulation with menotrophins, while 19 out of 91 women (21%) aged 41-45 years converted to optimal linings. Twenty-two out of 47 women (44%) who failed to develop optimal endometrial linings following ovarian stimulation converted to Grade IIA during subsequent cycles of exogenous oestrogen replacement. The financial, emotional, and physical burden associated with IVF/ET demands that patients with poor endometrial linings following ovarian stimulation with menotrophins be counselled with regard to either cancelling their cycles of treatment, or having their embryos cryopreserved for transfer to the uterus during a subsequent hormonal replacement cycle.

Assessment of the late proliferative phase endometrium by ultrasonography in patients undergoing in-vitro fertilization and embryo transfer (IVF/ET).

This study reports on 330 women aged 29 to 45 years, who underwent 411 cycles of in-vitro fertilization and embryo transfer (IVF/ET). Vaginal sonograms were performed during the late proliferative phase of natural cycles and cycles of controlled ovarian hyperstimulation (COH) with gonadotrophins, to evaluate both the thickness and echogenicity of the endometrium. Findings classified as Grade I; characterized by homogeneous echogenicity of the endometrium, and Grade II; characterized by an outer peripheral layer of dense echogenicity surrounding a central sonolucent area (i.e. a 'halo pattern'). Grades I and II were subclassified on the basis of thickness into A (greater than or equal to 9 mm) and B (less than 9 mm). Grade IIA ('optimal') was associated with a clinical pregnancy rate per embryo transfer of 33% while Grades IA, IB and IIB ('poor') were associated with a rate of only 7%. Women aged 41-45 years experienced a 25% incidence of 'poor' sonographic grades while the incidence in women less than or equal to 40 years of age was 5%. The presence of various uterine pathologies was associated with 'poor' endometrial grades in 86% of cases while only 11% of normal uteri manifested 'poor' grades. 'Optimal' endometrial grades in natural cycles were consistently associated with 'optimal' grades in ensuing cycles of COH (96%). Women with 'poor' endometrial grades in natural cycles improved in 55% of cases during subsequent COH. The results of this study indicate that sonographic assessment of the endometrial lining in the late proliferative phase during both natural and COH cycles is a valuable method for screening and managing IVF/ET candidates.